Novel insect inhibitory proteins

ABSTRACT

Pesticidal proteins exhibiting toxic activity against Lepidopteran pest species are disclosed, and include, but are not limited to, TIC7941, TIC7941PL_1, TIC7941PL_2, and TIC7941PL_3. DNA constructs are provided which contain a recombinant nucleic acid sequence encoding one or more of the disclosed pesticidal proteins. Transgenic plants, plant cells, seed, and plant parts resistant to Lepidopteran infestation are provided which contain recombinant nucleic acid sequences encoding the pesticidal proteins of the present invention. Methods for detecting the presence of the recombinant nucleic acid sequences or the proteins of the present invention in a biological sample, and methods of controlling Lepidopteran species pests using any of the TIC7941, TIC7941PL_1, TIC7941PL_2, and TIC7941PL_3 pesticidal proteins are also provided. Also disclosed are methods and compositions to improve the insecticidal activity of a pesticidal protein against an insect pest species. Further disclosed are method and compositions to reduce expression of a pesticidal protein in the reproductive tissues of a transgenic plant.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.62/795,066, filed Jan. 22, 2019, which is herein incorporated byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The file named “MONS469US_ST25.txt” containing a computer-readable formof the Sequence Listing was created on Jan. 21, 2020. This file is84,760 bytes (measured in MS-Windows®), filed contemporaneously byelectronic submission (using the United States Patent Office EFS-Webfiling system), and incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to the field of insect inhibitoryproteins. A novel class of proteins exhibiting insect inhibitoryactivity against agriculturally-relevant pests of crop plants and seedsare disclosed. In particular, the disclosed class of proteins isinsecticidally active against agriculturally-relevant pests of cropplants and seeds, particularly Lepidopteran species of insect pests.Plants, plant parts, and seeds containing a recombinant polynucleotideconstruct encoding one or more of the disclosed toxin proteins areprovided.

BACKGROUND OF THE INVENTION

Improving crop yield from agriculturally significant plants including,among others, corn, soybean, sugarcane, rice, wheat, vegetables, andcotton, has become increasingly important. In addition to the growingneed for agricultural products to feed, clothe and provide energy for agrowing human population, climate-related effects and pressure from thegrowing population to use land other than for agricultural practices arepredicted to reduce the amount of arable land available for farming.These factors have led to grim forecasts of food security, particularlyin the absence of major improvements in plant biotechnology andagronomic practices. In light of these pressures, environmentallysustainable improvements in technology, agricultural techniques, andpest management are vital tools to expand crop production on the limitedamount of arable land available for farming.

Insects, particularly insects within the order Lepidoptera andColeoptera, are considered a major cause of damage to field crops,thereby decreasing crop yields over infested areas. Lepidopteran pestspecies which negatively impact agriculture include, but are not limitedto, Black armyworm (Spodoptera exempta), Black cutworm (Agrotisipsilon), Corn earworm (Helicoverpa zea), Cotton leaf worm (Alabamaargillacea), Diamondback moth (Plutella xylostella), European corn borer(Ostrinia nubilalis), Fall armyworm (Spodoptera frugiperda), Cry1Fa1resistant Fall armyworm (Spodoptera frugiperda), Old World bollworm(OWB, Helicoverpa armigera), Southern armyworm (Spodoptera eridania),Soybean looper (Chrysodeixis includens), Spotted bollworm (Eariasvittella), Southwestern corn borer (Diatraea grandiosella), Tobaccobudworm (Heliothis virescens), Tobacco cutworm (Spodoptera litura, alsoknown as cluster caterpillar), Western bean cutworm (Striacostaalbicosta), and Velvet bean caterpillar (Anticarsia gemmatalis).

Historically, the intensive application of synthetic chemicalinsecticides was relied upon as the pest control agent in agriculture.Concerns for the environment and human health, in addition to emergingresistance issues, stimulated the research and development of biologicalpesticides. This research effort led to the progressive discovery anduse of various entomopathogenic microbial species, including bacteria.

The biological control paradigm shifted when the potential ofentomopathogenic bacteria, especially bacteria belonging to the genusBacillus, was discovered and developed as a biological pest controlagent. Strains of the bacterium Bacillus thuringiensis (Bt) have beenused as a source for pesticidal proteins since it was discovered that Btstrains show a high toxicity against specific insects. Bt strains areknown to produce delta-endotoxins that are localized within parasporalcrystalline inclusion bodies at the onset of sporulation and during thestationary growth phase (e.g., Cry proteins), and are also known toproduce secreted insecticidal protein. Upon ingestion by a susceptibleinsect, delta-endotoxins as well as secreted toxins exert their effectsat the surface of the midgut epithelium, disrupting the cell membrane,leading to cell disruption and death. Genes encoding insecticidalproteins have also been identified in bacterial species other than Bt,including other Bacillus and a diversity of additional bacterialspecies, such as Brevibacillus laterosporus, Lysinibacillus sphaericus(“Ls” formerly known as Bacillus sphaericus), Paenibacillus popilliaeand Paenibacillus lentimorbus.

Crystalline and secreted soluble insecticidal toxins are highly specificfor their hosts and have gained worldwide acceptance as alternatives tochemical insecticides. For example, insecticidal toxin proteins havebeen employed in various agricultural applications to protectagriculturally important plants from insect infestations, decrease theneed for chemical pesticide applications, and increase yields.Insecticidal toxin proteins are used to control agriculturally-relevantpests of crop plants by mechanical methods, such as spraying to dispersemicrobial formulations containing various bacteria strains onto plantsurfaces, and by using genetic transformation techniques to producetransgenic plants and seeds expressing insecticidal toxin protein.

The use of transgenic plants expressing insecticidal toxin proteins hasbeen globally adapted. For example, in 2016, 23.1 million hectares wereplanted with transgenic crops expressing Bt toxins and 75.4 millionhectares were planted with transgenic crops expressing Bt toxins stackedwith herbicide tolerance traits (ISAAA. 2016. Global Status ofCommercialized BiotecWGM Crops: 2016. ISAAA Brief No. 52. ISAAA: Ithaca,N.Y.). The global use of transgenic insect-protected crops and thelimited number of insecticidal toxin proteins used in these crops hascreated a selection pressure for existing insect alleles that impartresistance to the currently-utilized insecticidal proteins.

The development of resistance in target pests to insecticidal toxinproteins creates the continuing need for discovery and development ofnew forms of insecticidal toxin proteins that are useful for managingthe increase in insect resistance to transgenic crops expressinginsecticidal toxin proteins. New protein toxins with improved efficacyand which exhibit control over a broader spectrum of susceptible insectspecies will reduce the number of surviving insects which can developresistance alleles. In addition, the use in one plant of two or moretransgenic insecticidal toxin proteins toxic to the same insect pest anddisplaying different modes of action reduces the probability ofresistance in any single target insect species.

Thus, the inventors disclose herein a novel protein toxin family fromPaenibacillus lentimorbus, along with similar toxin proteins, variantproteins, and exemplary recombinant proteins that exhibit insecticidalactivity against target Lepidopteran species.

SUMMARY OF THE INVENTION

Disclosed herein is a novel group of pesticidal proteins with insectinhibitory activity (toxin proteins), referred to herein as TIC7941belonging to the TIC7941 protein toxin class, which are shown to exhibitinhibitory activity against one or more pests of crop plants. TheTIC7941 protein and proteins in the TIC7941 protein toxin class can beused alone or in combination with other insecticidal proteins and toxicagents in formulations and in planta, thus providing alternatives toinsecticidal proteins and insecticide chemistries currently in use inagricultural systems.

In one embodiment, disclosed in this application is a recombinantnucleic acid molecule comprising a heterologous promoter fragmentoperably linked to a polynucleotide segment encoding a pesticidalprotein or fragment thereof, wherein (a) said pesticidal proteincomprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2, SEQ IDNO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14; or (b)said pesticidal protein comprises an amino acid sequence having at least80% or, 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acidsequence identity to SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6; SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14; or (c) said polynucleotidesegment hybridizes to a polynucleotide having the nucleotide sequence ofSEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, or SEQ ID NO:13; or (d) said polynucleotide segment encoding apesticidal protein or fragment thereof comprises a polynucleotidesequence having at least 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or95%, or 98%, or 99%, or about 100% sequence identity to the nucleotidesequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, or SEQ ID NO:13; or (e) said recombinant nucleicacid molecule is in operable linkage with a vector, and said vector isselected from the group consisting of a plasmid, phagemid, bacmid,cosmid, and a bacterial or yeast artificial chromosome. The recombinantnucleic acid molecule can comprise a sequence that functions to expressthe pesticidal protein in a plant; or is expressed in a plant cell toproduce a pesticidally effective amount of pesticidal protein.

In another embodiment of this application are host cells comprising arecombinant nucleic acid molecule of the application, wherein the hostcell is selected from the group consisting of a bacterial and a plantcell. Contemplated bacterial host cells include Agrobacterium,Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas,Klebsiella, Pantoec, and Erwinia. In certain embodiments, said Bacillusspecies is Bacillus cereus or Bacillus thuringiensis, said Brevibacillusis Brevibacillus laterosperous, or Escherichia is Escherichia coli.Contemplated plant host cells include a dicotyledonous plant cell and amonocotyledonous plant cell. Contemplated plant cells further include analfalfa, banana, barley, bean, broccoli, cabbage, brassica, carrot,cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, citrus,coconut, coffee, corn, clover, cotton (Gossypium sp.), a cucurbit,cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, hops,leek, lettuce, Loblolly pine, millets, melons, nut, oat, olive, onion,ornamental, palm, pasture grass, pea, peanut, pepper, pigeonpea, pine,potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice,rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweetcorn, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato,triticale, turf grass, watermelon, and wheat plant cell.

In another embodiment, the pesticidal protein exhibits activity againstLepidopteran insects, including Velvet bean caterpillar, Sugarcaneborer, Lesser cornstalk borer, Corn earworm, Tobacco budworm, Soybeanlooper, Black armyworm, Southern armyworm, Fall armyworm, Beet armyworm,Old World bollworm, Oriental leaf worm, Pink bollworm, Black cutworm,Southwestern Corn Borer, Cotton leaf worm, Diamond back moth, Spottedboll worm, Tobacco cut worm, Western bean cutworm, and European cornborer.

Also contemplated in this application are plants comprising arecombinant nucleic acid molecule comprising a heterologous promoterfragment operably linked to a polynucleotide segment encoding apesticidal protein or fragment thereof, wherein: (a) said pesticidalprotein comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:2,SEQ ID NO:12, or SEQ ID NO:14; or (b) said pesticidal protein comprisesan amino acid sequence having at least 80% or, 85%, or 90%, or 95%, or98%, or 99%, or about 100% amino acid sequence identity to SEQ ID NO:4,SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:14; or (c) said polynucleotidesegment hybridizes under stringent hybridization conditions to thecompliment of the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:11, orSEQ ID NO:13; or (d) said plant exhibits a detectable amount of saidpesticidal protein. In certain embodiments, the pesticidal proteincomprises SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:14. Inone embodiment, the plant is either a dicotyledonous plant or amonocotyledonous plant. In another embodiment, the plant is furtherselected from the group consisting of an alfalfa, banana, barley, bean,broccoli, cabbage, brassica, carrot, cassava, castor, cauliflower,celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn,clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus,flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets,melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea,peanut, pepper, pigeon pea, pine, potato, poplar, pumpkin, Radiata pine,radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum,Southern pine, soybean, spinach, squash, strawberry, sugar beet,sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass,tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat.

In further embodiments, seeds comprising the recombinant nucleic acidmolecules are disclosed.

In another embodiment, an insect inhibitory composition comprising therecombinant nucleic acid molecules disclosed in this application armcontemplated. The insect inhibitory composition can further comprise anucleotide sequence encoding at least one other pesticidal agent that isdifferent from said pesticidal protein. In certain embodiments, the atleast one other pesticidal agent is selected from the group consistingof an insect inhibitory protein, an insect inhibitory dsRNA molecule,and an ancillary protein. It is also contemplated that the at least oneother pesticidal agent in the insect inhibitory composition exhibitsactivity against one or more pest species of the orders Lepidoptera,Coleoptera, or Hemiptera. The at least one other pesticidal agent in theinsect inhibitory composition is in one embodiment selected from thegroup consisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B,Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras, Cry1G,Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3Avariants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35,Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70, TIC400,TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900, TIC901,TIC1201, TIC1415, TIC3131, TIC2160, VIP3A, VIP3B, VIP3Ab, AXMI-001,AXMI-002, AXMI-030, AXMI-035, AXMI-036, AXMI-045, Axmi52, Axmi58,Axmi88, Axmi97, Axmi102, Axmi112, Axmi117, Axmi100, AXMI-115, AXMI-113,and AXMI-005, AXMI134, AXMI-150, Axmi171, AXMI-184, axmi196, axmi204,axmi207, axmi209, Axmi205, AXMI218, AXMI220, AXMI221z, AXMI222z,AXMI223z, AXMI224z and AXMI225z, AXMI238, AXMI270, AXMI279, AXMI335,AXMI345, AXMI-R1, and variants thereof, IP3 and variants thereof, DIG-3,DIG-5, DIG-10, DIG-11, DIG-657 protein, PHI-4 variants, PIP-72 variants,PIP-45 variants, PIP-64 variants, PIP-74 variants, PIP-77 variants,DIG-305, PIP-47 variants, DIG-17, DIG-90, DIG-79, and DIG-303.

Commodity products comprising a detectable amount of the recombinantnucleic acid molecules disclosed in this application are alsocontemplated. Such commodity products include commodity corn bagged by agrain handler, corn flakes, corn cakes, corn flour, corn meal, cornsyrup, corn oil, corn silage, corn starch, corn cereal, and the like,and corresponding soybean, rice, wheat, sorghum, pigeon pea, peanut,fruit, melon, and vegetable commodity products including, whereapplicable, juices, concentrates, jams, jellies, marmalades, and otheredible forms of such commodity products containing a detectable amountof such polynucleotides and or polypeptides of this application, wholeor processed cotton seed, cotton oil, lint, seeds and plant partsprocessed for feed or food, fiber, paper, biomasses, and fuel productssuch as fuel derived from cotton oil or pellets derived from cotton ginwaste, whole or processed soybean seed, soybean oil, soybean protein,soybean meal, soybean flour, soybean flakes, soybean bran, soybean milk,soybean cheese, soybean wine, animal feed comprising soybean, papercomprising soybean, cream comprising soybean, soybean biomass, and fuelproducts produced using soybean plants and soybean plant parts.

Also contemplated in this application is a method of producing seedcomprising the recombinant nucleic acid molecules disclosed in thisapplication. The method comprises planting at least one of the seedcomprising the recombinant nucleic acid molecules disclosed in thisapplication; growing plant from the seed; and harvesting seed from theplants, wherein the harvested seed comprises the recombinant nucleicacid molecules in this application.

In another illustrative embodiment, a plant resistant to insectinfestation, is provided wherein the cells of said plant comprise: (a) arecombinant nucleic acid molecule encoding an insecticidally effectiveamount of a pesticidal protein as set forth in SEQ ID NO:4, SEQ ID NO:2,SEQ ID NO:12, or SEQ ID NO:14; or (b) an insecticidally effective amountof a protein comprising an amino acid sequence having at least 80% or,85%, or 90%, or 95%, or about 100% amino acid sequence identity to SEQID NO:4, SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:14.

Also disclosed in this application are methods for controlling aLepidopteran species pest, and controlling a Lepidopteran species pestinfestation of a plant, particularly a crop plant. The method comprises,in one embodiment, (a) contacting the pest with an insecticidallyeffective amount of a pesticidal proteins as set forth in SEQ ID NO:4,SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:14; or (b) contacting the pestwith an insecticidally effective amount of one or more pesticidalproteins comprising an amino acid sequence having at least 80%, 85%, or90%, or 95%, or about 100% amino acid sequence identity to identity toSEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:14.

Further provided herein is a method of detecting the presence of arecombinant nucleic acid molecule comprising a polynucleotide segmentencoding a pesticidal protein or fragment thereof, wherein: (a) saidpesticidal protein comprises the amino acid sequence of SEQ ID NO:4, SEQID NO:2, SEQ ID NO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ IDNO:14; or (b) said pesticidal protein comprises an amino acid sequencehaving at least 80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about100% amino acid sequence identity to SEQ ID NO:4, SEQ ID NO:2, SEQ IDNO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14; or (c)said polynucleotide segment hybridizes to a polynucleotide having thenucleotide sequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5; SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO:13. In one embodiment ofthe invention, the method comprises contacting a sample of nucleic acidswith a nucleic acid probe that hybridizes under stringent hybridizationconditions with genomic DNA from a plant comprising a polynucleotidesegment encoding a pesticidal protein or fragment thereof providedherein, and does not hybridize under such hybridization conditions withgenomic DNA from an otherwise isogenic plant that does not comprise thesegment, wherein the probe is homologous or complementary to SEQ IDNO:3, SEQ ID NO:11, or SEQ ID NO:13, or a sequence that encodes apesticidal protein comprising an amino acid sequence having at least80%, or 85%, or 90%, or 95%, or 98%, or 99%, or about 100% amino acidsequence identity to SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:12, or SEQ IDNO:14. The method may further comprise (a) subjecting the sample andprobe to stringent hybridization conditions; and (b) detectinghybridization of the probe with DNA of the sample.

Also provided by the invention are methods of detecting the presence ofa pesticidal protein or fragment thereof in a sample comprising protein,wherein said pesticidal protein comprises the amino acid sequence of SEQID NO:4, SEQ ID NO:2, SEQ ID NO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, or SEQ ID NO:14; or said pesticidal protein comprises an aminoacid sequence having at least 80%, or 85%, or 90%, or 95%, or 98%, or99%, or about 100% amino acid sequence identity to SEQ ID NO:4, SEQ IDNO:2, SEQ ID NO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ IDNO:14. In one embodiment, the method comprises: (a) contacting a samplewith an immunoreactive antibody; and (b) detecting the presence of theprotein. In some embodiments the step of detecting comprises an ELISA,or a Western blot.

Also disclosed in this application is a method for improving theinsecticidal activity of a native insecticidal protein against an insectpest species, comprising: engineering a variant insecticidal protein byinserting a DNA fragment encoding an insect gut receptor binding peptideinto a coding sequence encoding the insecticidal protein; wherein theinsecticidal activity of the engineered insecticidal protein is greaterthan the insecticidal activity of the native insecticidal protein tosaid insect pest species. In one embodiment of the invention, the insectgut receptor can be a cadherin-like protein (CADR), a GPI-anchoredaminopeptidase-N (APN), a GPI-anchored alkaline phosphatase, atransmembrane ABC transporter, or an ADAM metalloprotease. In anotherembodiment of the invention, the DNA fragment encoding an insect gutreceptor binding peptide is selected from the group consisting of SEQ IDNO:15 and SEQ ID NO:16 and encodes the receptor binding peptide providedas SEQ ID NO:17.

In an embodiment of the invention are recombinant nucleic acid moleculecomprising a heterologous promoter operably linked to a polynucleotidesegment encoding a pesticidal protein or pesticidal fragment thereof,operably linked to a DNA sequence comprising a reproductivetissue-specific miRNA target binding site element, wherein said miRNAtarget binding site element is heterologous with respect to saidpolynucleotide segment encoding a pesticidal protein or pesticidalfragment thereof. The miRNA target binding site elements are selectedfrom the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.

In yet another embodiment of the invention is a method for reducingexpression of a pesticidal protein in the reproductive tissue of atransgenic plant, comprising expressing in said transgenic plant arecombinant nucleic acid molecule comprising a heterologous promoteroperably linked to a polynucleotide segment encoding a pesticidalprotein or pesticidal fragment thereof, operably linked to a DNAsequence comprising a reproductive tissue-specific miRNA target bindingsite element, wherein said miRNA target binding site element isheterologous with respect to said polynucleotide segment encoding apesticidal protein or pesticidal fragment thereof. The miRNA targetbinding site elements are selected from the group consisting of SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQID NO:23. A further embodiment of the invention is a recombinant DNAmolecule selected from the group consisting of SEQ ID NO:25 and SEQ IDNO:26.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleic acid sequence encoding a TIC7941 pesticidalprotein obtained from Paenibacillus lentimorbus species DSC020651.

SEQ ID NO:2 is the amino acid sequence of the TIC7941 pesticidalprotein.

SEQ ID NO:3 is a synthetic coding sequence encoding a TIC7941PL_1pesticidal protein designed for expression in a plant cell.

SEQ ID NO:4 is the amino acid sequence of the TIC7941PL_1 proteinwherein an additional alanine amino acid is inserted immediatelyfollowing the initiating methionine.

SEQ ID NO:5 is a nucleic acid sequence encoding a TIC7941_His pesticidalprotein, wherein a nucleic acid sequence encoding a Histidine tag isoperably linked 5′ and in frame to the TIC7941 coding sequence.

SEQ ID NO:6 is the amino acid sequence of the TIC7941_His pesticidalprotein.

SEQ ID NO:7 is a nucleic acid sequence encoding a TIC7941_2Hispesticidal protein, wherein a nucleic acid sequence encoding a Histidinetag is operably linked 5′ and in frame.

SEQ ID NO:8 is the amino acid sequence of the TIC7941_2His pesticidalprotein.

SEQ ID NO:9 is a nucleic acid sequence encoding a TIC7941_3Hispesticidal protein, wherein a nucleic acid sequence encoding a Histidinetag is operably linked 5′ and in frame.

SEQ ID NO:10 is the amino acid sequence of the TIC7941_3His pesticidalprotein.

SEQ ID NO:11 is a synthetic coding sequence encoding a TIC7941PL_2pesticidal protein designed for expression in a plant cell.

SEQ ID NO:12 is the amino acid sequence of TIC7941PL_2 wherein anadditional alanine amino acid is inserted immediately following theinitiating methionine.

SEQ ID NO:13 is a synthetic coding sequence encoding a TIC7941PL_3pesticidal protein designed for expression in a plant cell.

SEQ ID NO:14 is the amino acid sequence of TIC7941PL_3 wherein anadditional alanine amino acid is inserted immediately following theinitiating methionine.

SEQ ID NO:15 is a synthetic coding sequence (FAWPEPBIN_Bac) encoding theFAW ABCc4 receptor binding peptide sequence FAWPEPBIN for expression inbacteria. The synthetic sequence is found within nucleotide positions2413-2448 of TIC7941_2His and within nucleotide positions 2410-2445 ofTIC7941_3His.

SEQ ID NO:16 is a synthetic coding sequence (FAWPEPBIN_PL) encoding theFAW ABCc4 receptor binding peptide sequence FAWPEPBIN for expression ina plant cell. The synthetic sequence is found within nucleotidepositions 2386-2421 of TIC7941PL_2 and within nucleotide positions2383-2418 of TIC7941PL_3.

SEQ ID NO:17 is the FAW ABCc4 receptor binding peptide sequence(FAWPEPBIN) encoded by SEQ ID NO:15 and SEQ ID NO:16 and is located atamino acid positions 805-816 of TIC7941_2His, 804-815 of TIC7941_3His,796-807 of TIC7941PL_2, and 795-806 of TIC7941PL_3.

SEQ ID NO:18 is a DNA sequence encoding an miRNA target binding siteGm.miR395_1.

SEQ ID NO:19 is a DNA sequence encoding an miRNA binding target siteGm.miR395_2.

SEQ ID NO:20 is a DNA sequence (SUP-miR395) wherein the miRNA targetbinding sites Gm.miR395_1 and Gm.miR395_2 are linked using a DNAsequence SP-ART.8a-1.

SEQ ID NO:21 is a DNA sequence encoding an miRNA target binding siteGm.miR4392_1.

SEQ ID NO:22 is a DNA sequence encoding an miRNA target binding siteGm.miR4392_2.

SEQ ID NO:23 is a DNA sequence (SUP-miR4392) wherein the miRNA targetbinding sites Gm.miR4392_1 and Gm.miR4392_2 are linked using a DNAsequence SP-ART.8a-1.

SEQ ID NO:24 is the DNA sequence of the linker SP-ART.8a-1.

SEQ ID NO:25 is a DNA sequence (TIC7941PL_1-mi395) encoding TIC7941PL_1operably linked to SUP-miR395.

SEQ ID NO:26 is a DNA sequence (TIC7941PL_1-mi4392) encoding TIC7941PL_1operably linked to SUP-miR4392.

DETAILED DESCRIPTION OF THE INVENTION

The problem in the art of agricultural pest control can be characterizedas a need for new toxin proteins that are efficacious against targetpests, exhibit broad spectrum toxicity against target pest species, arecapable of being expressed in plants without causing undesirableagronomic issues, and provide an alternative mode of action compared tocurrent toxins that are used commercially in plants.

Novel pesticidal proteins exemplified by TIC7941, TIC7941PL_1,TIC7941PL_2, and TIC7941PL_3 are disclosed herein, and address each ofthese needs, particularly against a broad spectrum of Lepidopteraninsect pests, and more particularly against Black cutworm (Agrotisipsilon), Corn earworm (Helicoverpa zea), European corn borer (Ostrinianubilalis), Fall armyworm (Spodoptera frugiperda), Southern armyworm(Spodoptera eridania), Soybean looper (Chrysodeixis includens),Southwestern corn borer (Diatraea grandiosella).

Reference in this application to TIC7941, “TIC7941 protein”, “TIC7941protein toxin”, “TIC7941 toxin protein”, “TIC7941 pesticidal protein”,“TIC7941-related toxins”, “TIC7941-related toxin proteins”, TIC7941PL_1,“TIC7941PL_1 protein”, “TIC7941PL_1 protein toxin”, “TIC7941PL_1 toxinprotein”, “TIC7941PL_1 pesticidal protein”, “TIC7941PL_1-relatedtoxins”, “TIC7941PL_1-related toxin proteins”, and the like, refer toany novel pesticidal protein or insect inhibitory protein, thatcomprises, that consists of, that is substantially homologous to, thatis similar to, or that is derived from any pesticidal protein or insectinhibitory protein sequence of TIC7941 (SEQ ID NO:2), TIC7941PL_1 (SEQID NO:4), TIC7941PL_2 (SEQ ID NO:12), and TIC7941PL_3 (SEQ ID NO:14) andpesticidal or insect inhibitory segments thereof, or combinationsthereof, that confer activity against Lepidopteran pests, including anyprotein exhibiting pesticidal or insect inhibitory activity if alignmentof such protein with TIC7941, TIC7941PL_1, TIC7941PL_2, or TIC7941PL_3results in amino acid sequence identity of any fraction percentage formabout 80% to about 100% percent. The TIC7941, TIC7941PL_1, TIC7941PL_2,and TIC7941PL_3 proteins include both the plastid-targeted andnon-plastid targeted form of the proteins.

The term “segment” or “fragment” is used in this application to describeconsecutive amino acid or nucleic acid sequences that are shorter thanthe complete amino acid or nucleic acid sequence describing a TIC7941protein. A segment or fragment exhibiting insect inhibitory activity isalso disclosed in this application if alignment of such segment orfragment, with the corresponding section of the TIC7941 protein setforth in SEQ ID NO:2, or TIC7941PL_1 protein set forth in SEQ ID NO:4,or TIC7941PL_2 protein set forth in SEQ ID NO:12, or TIC7941PL_3 proteinset forth as SEQ ID NO:14 results in amino acid sequence identity of anyfraction percentage from about 80 to about 100 percent between thesegment or fragment and the corresponding section of the TIC7941,TIC7941PL_1, TIC7941PL_2, or TIC7941PL_3 protein.

In still further specific embodiments, a fragment of a TIC7941,TIC7941PL_1, TIC7941PL_2, or TIC7941PL_3 protein may be defined asexhibiting pesticidal activity possessed by the starting proteinmolecule from which it is derived. A fragment of a nucleic acid sequenceencoding a TIC7941, TIC7941PL_1, TIC7941PL_2, or TIC7941PL_3 protein maybe defined as encoding a protein exhibiting the pesticidal activitypossessed by the protein molecule encoded by the starting nucleic acidsequence from which it is derived. A fragment or variant describedherein may further comprise a domain identified herein which isresponsible for the pesticidal activity of a protein.

In specific embodiments, fragments of a TIC7941, TIC7941PL_1,TIC7941PL_2, or TIC7941PL_3 protein are provided comprising at leastabout 50, at least about 75, at least about 95, at least about 100, atleast about 125, at least about 150, at least about 175, at least about200, at least about 225, at least about 250, at least about 275, atleast about 300, at least about 500, at least about 600, at least about700, at least about 750, at least about 800, at least about 900, atleast about 1000, at least about 1100, at least about 1150, or at leastabout 1175 contiguous amino acids, or longer, of a TIC7941, TIC7941PL_1,TIC7941PL_2, or TIC7941PL_3 protein having pesticidal activity asdisclosed herein. In certain embodiments, the invention providesfragments of any one of SEQ ID NOs: 2, 4, 12, or 14, having the activityof the full length sequence. Methods for producing such fragments from astarting molecule are well known in the art.

Reference in this application to the terms “active” or “activity”,“pesticidal activity” or “pesticidal” or “insecticidal activity”,“insect inhibitory” or “insecticidal” refer to efficacy of a toxicagent, such as a protein toxin, in inhibiting (inhibiting growth,feeding, fecundity, or viability), suppressing (suppressing growth,feeding, fecundity, or viability), controlling (controlling the pestinfestation, controlling the pest feeding activities on a particularcrop containing an effective amount of the TIC7941 protein) or killing(causing the morbidity, mortality, or reduced fecundity of) a pest.These terms are intended to include the result of providing apesticidally effective amount of a toxic protein to a pest where theexposure of the pest to the toxic protein results in morbidity,mortality, reduced fecundity, or stunting. These terms also includerepulsion of the pest from the plant, a tissue of the plant, a plantpart, seed, plant cells, or from the particular geographic locationwhere the plant may be growing, as a result of providing a pesticidallyeffective amount of the toxic protein in or on the plant. In general,pesticidal activity refers to the ability of a toxic protein to beeffective in inhibiting the growth, development, viability, feedingbehavior, mating behavior, fecundity, or any measurable decrease in theadverse effects caused by an insect feeding on this protein, proteinfragment, protein segment or polynucleotide of a particular target pest,including but not limited to insects of the order Lepidoptera. The toxicprotein can be produced by the plant or can be applied to the plant orto the environment within the location where the plant is located. Theterms “bioactivity”, “effective”, “efficacious” or variations thereofare also terms interchangeably utilized in this application to describethe effects of proteins of the present invention on target insect pests.

A pesticidally effective amount of a toxic agent, when provided in thediet of a target pest, exhibits pesticidal activity when the toxic agentcontacts the pest. A toxic agent can be a pesticidal protein or one ormore chemical agents known in the art. Pesticidal or insecticidalchemical agents and pesticidal or insecticidal protein agents can beused alone or in combinations with each other. Chemical agents includebut are not limited to dsRNA molecules targeting specific genes forsuppression in a target pest, organochlorides, organophosphates,carbamates, pyrethroids, neonicotinoids, and ryanoids. Pesticidal orinsecticidal protein agents include the protein toxins set forth in thisapplication, as well as other proteinaceous toxic agents including thosethat target Lepidopterans, as well as protein toxins that are used tocontrol other plant pests such as Cry and Cyt proteins available in theart for use in controlling Coleopteran, Hemipteran and Homopteranspecies.

It is intended that reference to a pest, particularly a pest of a cropplant, means insect pests of crop plants, particularly those Lepidopterainsect pests that are controlled by the TIC7941 protein toxin class.However, reference to a pest can also include Coleopteran, Hemipteranand Homopteran insect pests of plants, as well as nematodes and fungiwhen toxic agents targeting these pests are co-localized or presenttogether with the TIC7941 protein or a protein that is 80 to about 100percent identical to TIC7941protein.

The TIC7941 proteins are related by a common function and exhibitinsecticidal activity towards insect pests from the Lepidoptera insectspecies, including adults, pupae, larvae, and neonates.

The insects of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers, and heliothines in the Family Noctuidae,e.g., Fall armyworm (Spodoptera frugiperda), Beet armyworm (Spodopteraexigua), Black armyworm (Spodoptera exempta), Southern armyworm(Spodoptera eridania), bertha armyworm (Mamestra configurata), blackcutworm (Agrotis ipsilon), cabbage looper (Trichoplusia ni), soybeanlooper (Pseudoplusia includens), velvetbean caterpillar (Anticarsiagemmatalis), green cloverworm (Hypena scabra), tobacco budworm(Heliothis virescens), granulate cutworm (Agrotis subterranea), armyworm(Pseudaletia unipuncta), western cutworm (Agrotis orthogonia); borers,casebearers, webworms, coneworms, cabbageworms and skeletonizers fromthe Family Pyralidae, e.g., European corn borer (Ostrinia nubilalis),navel orangeworm (Amyelois transitella), corn root webworm (Crambuscaliginosellus), sod webworm (Herpetogramma licarsisalis), sunflowermoth (Homoeosoma electellum), lesser cornstalk borer (Elasmopalpuslignosellus); leafrollers, budworms, seed worms, and fruit worms in theFamily Tortricidae, e.g., codling moth (Cydia pomonella), grape berrymoth (Endopiza viteana), oriental fruit moth (Grapholita molesta),sunflower bud moth (Suleima helianthana); and many other economicallyimportant Lepidoptera, e.g., diamondback moth (Plutella xylostella),pink bollworm (Pectinophora gossypiella), and gypsy moth (Lymantriadispar). Other insect pests of order Lepidoptera include, e.g., cottonleaf worm (Alabama argillacea), fruit tree leaf roller (Archipsargyrospila), European leafroller (Archips rosana) and other Archipsspecies, (Chilo suppressalis, Asiatic rice borer, or rice stem borer),rice leaf roller (Cnaphalocrocis medinalis), corn root webworm (Crambuscaliginosellus), bluegrass webworm (Crambus teterrellus), southwesterncorn borer (Diatraea grandiosella), surgarcane borer (Diatraeasaccharalis), spiny bollworm (Earias insulana), spotted bollworm (Eariasvittella), American bollworm (Helicoverpa armigera), corn earworm(Helicoverpa zea, also known as soybean podworm and cotton bollworm),tobacco budworm (Heliothis virescens), sod webworm (Herpetogrammalicarsisalis), Western bean cutworm (Striacosta albicosta), Europeangrape vine moth (Lobesia botrana), citrus leafminer (Phyllocnistiscitrella), large white butterfly (Pieris brassicae), small whitebutterfly (Pieris rapae, also known as imported cabbageworm), beetarmyworm (Spodoptera exigua), tobacco cutworm (Spodoptera litura, alsoknown as cluster caterpillar), and tomato leafminer (Tuta absoluta).

Reference in this application to an “isolated DNA molecule”, or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. Similarly, a nucleotide sequence encoding an insecticidal proteinor any naturally occurring insecticidal variant of that protein would bean isolated nucleotide sequence so long as the nucleotide sequence wasnot within the DNA of the bacterium from which the sequence encoding theprotein is naturally found. A synthetic nucleotide sequence encoding theamino acid sequence of the naturally occurring insecticidal proteinwould be considered to be isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of a plant or bacterium, or present in an extrachromosomal vector,would be considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium.

As used herein, a “recombinant DNA molecule” is a DNA moleculecomprising a combination of DNA molecules that would not naturally occurtogether without human intervention. For instance, a recombinant DNAmolecule may be a DNA molecule that is comprised of at least two DNAmolecules heterologous with respect to each other, a DNA molecule thatcomprises a DNA sequence that deviates from DNA sequences that exist innature, or a DNA molecule that has been incorporated into a host cell'sDNA by genetic transformation or gene editing. Similarly, a “recombinantprotein molecule” is a protein molecule comprising a combination ofamino acids that would not naturally occur together without humanintervention. For example, a recombinant protein molecule may be aprotein molecule that is comprised of at least two amino acid moleculesheterologous with respect to each other, a protein molecule thatcomprises an amino acid sequence that deviates from amino acid sequencesthat exist in nature, or a protein molecule that is expressed in a hostcell as a result of genetic transformation of the host cell or by geneediting of the host cell genome.

As described further in this application, an open reading frame (ORF)encoding TIC7941 (SEQ ID NO:2) was discovered in DNA obtained fromPaenibacillus lentimorbus strain DSC020651. The coding sequence wascloned and expressed in microbial host cells to produce recombinantproteins used in bioassays. Bioassay using microbial host cell-derivedproteins of TIC7941 demonstrated activity against the Lepidopteranspecies Black cutworm (Agrodis ipsilon), Corn earworm (Helicoverpa zea),European corn borer (Ostrinia nubilalis), Southern armyworm (Spodopteraeridania), Soybean looper (Chrysodeixis includens), and Southwesterncorn borer (Diatraea grandiosella).

Synthetic sequences encoding TIC7941 and variants of TIC7941 weredesigned for expression in a plant cell. The coding sequence,TIC7941PL_1 (SEQ ID NO:3) encodes the TIC7941PL_1 insecticidal proteinwhich is identical to the TIC7941 protein sequence with the exception ofan additional alanine amino acid inserted after the initiatingmethionine to improve expression. When expressed in transgenic corn,TIC7941PL_1 demonstrated insecticidal activity against Black cutworm(BCW, Agrotis ipsilon), Corn earworm (CEW, Helicoverpa zea), andSouthwestern corn borer (SWCB, Diatraea grandiosella) in leaf discassays. When expressed in transgenic soybean plants, TIC7941PL_1demonstrates insecticidal activity against Southern armyworm (SAW,Spodoptera eridania), Soybean looper (SBL, Chrysodeixis includens), andSoybean podworm (SPW, Helicoverpa zea) in leaf disc assays. TheTIC7941PL_2 coding sequence (SEQ ID NO:11) and TIC7941PL_3 codingsequence (SEQ ID NO:13) encode the TIC7941PL_2 (SEQ ID NO:12) andTIC7941PL_3 (SEQ ID NO:14) insecticidal proteins, respectively. Theycontain an additional alanine amino acid inserted after the initiatingmethionine to improve expression. Both TIC7941PL_2 and TIC7941PL_3 alsocontain a Fall armyworm transmembrane ABC transporter (ABCc4) proteinbinding peptide fragment inserted within the domain 2 loop of TIC7941.In TIC7941PL_2 the ABCc4 protein binding fragment is located at aminoacid positions 796-807. In TIC7941PL_3the ABCc4 protein binding fragmentis located at amino acid positions 795-806.

For expression in plant cells, the TIC7941PL_1, TIC7941PL_2, orTIC7941PL_3 protein can be expressed to reside in the cytosol ortargeted to various organelles of the plant cell. For example, targetinga protein to the chloroplast may result in increased levels of expressedprotein in a transgenic plant while preventing off-phenotypes fromoccurring. Targeting may also result in an increase in pest resistanceefficacy in the transgenic event. A target peptide or transit peptide isa short (3-70 amino acids long) peptide chain that directs the transportof a protein to a specific region in the cell, including the nucleus,mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast,peroxisome and plasma membrane. Some target peptides are cleaved fromthe protein by signal peptidases after the proteins are transported. Fortargeting to the chloroplast, proteins contain transit peptides whichare around 40-50 amino acids. For descriptions of the use of chloroplasttransit peptides, see U.S. Pat. Nos. 5,188,642 and 5,728,925. Manychloroplast-localized proteins are expressed from nuclear genes asprecursors and are targeted to the chloroplast by a chloroplast transitpeptide (CTP). Examples of such isolated chloroplast proteins include,but are not limited to, those associated with the small subunit (SSU) ofribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxinoxidoreductase, the light-harvesting complex protein I and protein II,thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS), andtransit peptides described in U.S. Pat. No. 7,193,133. It has beendemonstrated in vivo and in vitro that non-chloroplast proteins may betargeted to the chloroplast by use of protein fusions with aheterologous CTP and that the CTP is sufficient to target a protein tothe chloroplast. Incorporation of a suitable chloroplast transit peptidesuch as the Arabidopsis thaliana EPSPS CTP (CTP2) (see, Klee et al.,Mol. Gen. Genet. 210:437-442, 1987) or the Petunia hybrida EPSPS CTP(CTP4) (see, della-Cioppa et al., Proc. Natl. Acad. Sci. USA83:6873-6877, 1986) has been shown to target heterologous EPSPS proteinsequences to chloroplasts in transgenic plants (see, U.S. Pat. Nos.5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP508909; and EP 924299). For targeting the TIC7941, TIC7941PL_1,TIC7941PL_2, or TIC7941PL_3 toxin protein to the chloroplast, a sequenceencoding a chloroplast transit peptide is placed 5′ in operable linkageand in frame to a synthetic coding sequence encoding the TIC7941,TIC7941PL_1, TIC7941PL_2, or TIC7941PL_3 toxin protein that has beendesigned for optimal expression in plant cells.

It is contemplated that additional toxin protein sequences related toTIC7941 can be created by using the amino acid sequence of TIC7941 tocreate novel proteins with novel properties. The TIC7941 toxin proteinscan be aligned to combine differences at the amino acid sequence levelinto novel amino acid sequence variants and making appropriate changesto the recombinant nucleic acid sequence encoding the variants.

This disclosure further contemplates that improved variants of theTIC7941 protein toxin class can be engineered in planta by using variousgene editing methods known in the art. Such technologies used for genomeediting include, but are not limited to, ZFN (zinc-finger nuclease),meganucleases, TALEN (Transcription activator-like effector nucleases),and CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR-associated) systems. These genome editing methodscan be used to alter the toxin protein coding sequence transformedwithin a plant cell to a different toxin coding sequence. Specifically,through these methods, one or more codons within the toxin codingsequence is altered to engineer a new protein amino acid sequence.Alternatively, a fragment within the coding sequence is replaced ordeleted, or additional DNA fragments are inserted into the codingsequence, to engineer a new toxin coding sequence. The new codingsequence can encode a toxin protein with new properties such asincreased activity or spectrum against insect pests, as well as provideactivity against an insect pest species wherein resistance has developedagainst the original insect toxin protein. The plant cell comprising thegene edited toxin coding sequence can be used by methods known in theart to generate whole plants expressing the new toxin protein.

It is also contemplated that fragments of TIC7941 or protein variantsthereof can be truncated forms wherein one or more amino acids aredeleted from the N-terminal end, C-terminal end, the middle of theprotein, or combinations thereof wherein the fragments and variantsretain insect inhibitory activity. These fragments can be naturallyoccurring or synthetic variants of TIC7941 or derived protein variants,but should retain the insect inhibitory activity of at least TIC7941. Afragment or variant described herein may further comprise a domainidentified herein which is responsible for the pesticidal activity of aprotein.

Proteins that resemble the proteins in the TIC7941 protein toxin classcan be identified and compared to each other using various computerbased algorithms known in the art (see Table 1). Amino acid sequenceidentities reported in this application are a result of a Clustal Walignment using these default parameters: Weight matrix: blosum, Gapopening penalty: 10.0, Gap extension penalty: 0.05, Hydrophilic gaps:On, Hydrophilic residues: GPSNDQERK, Residue-specific gap penalties: On(Thompson, et al (1994) Nucleic Acids Research, 22:4673-4680). Percentamino acid identity is further calculated by the product of 100%multiplied by (amino acid identities/length of subject protein). Otheralignment algorithms are also available in the art and provide resultssimilar to those obtained using a Clustal W alignment and arecontemplated herein.

It is intended that a protein exhibiting insect inhibitory activityagainst a Lepidopteran insect species is related to a member of theTIC7941 protein toxin class if the protein is used in a query, e.g., ina Clustal W alignment, and the proteins of the present invention as setforth as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:10 areidentified as hits in such alignment in which the query protein exhibitsat least 80% to about 100% amino acid identity along the length of thequery protein that is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or anyfraction percentage in this range.

In addition to percent identity, TIC7941 proteins can also be related byprimary structure (conserved amino acid motifs), by length (about 807amino acids), and by other characteristics. Characteristics of theTIC7941 protein toxins are reported in Table 1.

TABLE 1 Selected characteristics of the TIC7941 protein toxin class. No.of No. of No. of Molecular Weight Amino Acid Isoelectric Charge atStrongly Basic (−) Strongly Acidic Hydrophobic No. of Polar Protein (inDaltons) Length Point PH 7.0 Amino Acids Amino Acids Amino Acids AminoAcids TIC7941 91187.48 807 4.4561 −35.5 87 118 394 413 TIC7941PL_191258.56 808 4.4561 −35.5 87 118 395 413 TIC7941PL_2 92245.74 817 4.4414−36.5 87 119 402 415 TIC7941PL_3 92203.70 817 4.4544 −35.5 87 118 402415

As described further in the Examples, synthetic nucleic acid moleculesequences encoding variants of TIC7941 were designed for use in plants.Exemplary recombinant nucleic acid molecule sequences that were designedfor use in plants encoding the TIC7941PL_1, TIC7941PL_2, and TIC7941PL_3proteins are presented as SEQ ID NO:3, SEQ ID NO:11, and SEQ ID NO:13,respectively. The TIC7941PL_1, TIC7941PL_2, and TIC7941PL_3 proteinshave an additional alanine amino acid immediately following theinitiating methionine relative to the TIC7941 protein. This additionalalanine residue is believed to improve expression of the protein inplanta. The TIC7941PL_2 and TIC7941PL_3 proteins also comprise the ABCc4peptide binding fragment to improve efficacy of the proteins againstFall armyworm (Spodoptera frugiperda).

Expression cassettes and vectors containing a recombinant nucleic acidmolecule sequence can be constructed and introduced into corn, soybeanor cotton plant cells in accordance with transformation methods andtechniques known in the art. For example, Agrobacterium-mediatedtransformation is described in U.S. Patent Application Publications2009/0138985A1 (soybean), 2008/0280361A1 (soybean), 2009/0142837A1(corn), 2008/0282432 (cotton), 2008/0256667 (cotton), 2003/0110531(wheat), 2001/0042257 A1 (sugar beet), U.S. Pat. No. 5,750,871 (canola),7,026,528 (wheat), and 6,365,807 (rice), and in Arencibia et al. (1998)Transgenic Res. 7:213-222 (sugarcane) all of which are incorporatedherein by reference in their entirety. Transformed cells can beregenerated into transformed plants that express TIC7941PL_1,TIC7941PL_2, or TIC7941PL_3 protein and demonstrate pesticidal activitythrough bioassays performed in the presence of Lepidopteran pest larvaeusing plant leaf disks obtained from the transformed plants. Plants canbe derived from the plant cells by regeneration, seed, pollen, ormeristem transformation techniques. Methods for transforming plants areknown in the art.

As an alternative to traditional transformation methods, a DNA sequence,such as a transgene, expression cassette(s), etc., may be inserted orintegrated into a specific site or locus within the genome of a plant orplant cell via site-directed integration. Recombinant DNA construct(s)and molecule(s) of this disclosure may thus include a donor templatesequence comprising at least one transgene, expression cassette, orother DNA sequence for insertion into the genome of the plant or plantcell. Such donor template for site-directed integration may furtherinclude one or two homology arms flanking an insertion sequence (i.e.,the sequence, transgene, cassette, etc., to be inserted into the plantgenome). The recombinant DNA construct(s) of this disclosure may furthercomprise an expression cassette(s) encoding a site-specific nucleaseand/or any associated protein(s) to carry out site-directed integration.These nuclease expressing cassette(s) may be present in the samemolecule or vector as the donor template (in cis) or on a separatemolecule or vector (in trans). Several methods for site-directedintegration are known in the art involving different proteins (orcomplexes of proteins and/or guide RNA) that cut the genomic DNA toproduce a double strand break (DSB) or nick at a desired genomic site orlocus. Briefly as understood in the art, during the process of repairingthe DSB or nick introduced by the nuclease enzyme, the donor templateDNA may become integrated into the genome at the site of the DSB ornick. The presence of the homology arm(s) in the donor template maypromote the adoption and targeting of the insertion sequence into theplant genome during the repair process through homologous recombination,although an insertion event may occur through non-homologous end joining(NHEJ). Examples of site-specific nucleases that may be used includezinc-finger nucleases, engineered or native meganucleases,TALE-endonucleases, and RNA-guided endonucleases (e.g., Cas9 or Cpf1).For methods using RNA-guided site-specific nucleases (e.g., Cas9 orCpf1), the recombinant DNA construct(s) will also comprise a sequenceencoding one or more guide RNAs to direct the nuclease to the desiredsite within the plant genome.

Recombinant nucleic acid molecule compositions that encode TIC7941proteins are contemplated. For example, TIC7941, TIC7941PL_1,TIC7941PL_2, and TIC7941PL_3 proteins can be expressed with recombinantDNA constructs in which a polynucleotide molecule with an ORF encodingthe protein is operably linked to genetic expression elements such as apromoter and any other regulatory element necessary for expression inthe system for which the construct is intended. Non-limiting examplesinclude a plant-functional promoter operably linked to a TIC7941 proteinencoding sequence for expression of the protein in plants or aBt-functional promoter operably linked to a TIC7941 protein encodingsequence for expression of the protein in a Bt bacterium or otherBacillus species. Other elements can be operably linked to the TIC7941protein encoding sequence including, but not limited to, enhancers,introns, untranslated leaders, encoded protein immobilization tags(HIS-tag), translocation peptides (i.e., plastid transit peptides,signal peptides), polypeptide sequences for post-translational modifyingenzymes, ribosomal binding sites, and RNAi target sites. Exemplaryrecombinant polynucleotide molecules provided herewith include, but arenot limited to, a heterologous promoter operably linked to apolynucleotide such as SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13 that encodes therespective polypeptides or proteins having the amino acid sequence asset forth in SEQ ID NO:4, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, and SEQ ID NO:14. A heterologous promoter can alsobe operably linked to synthetic DNA coding sequences encoding a plastidtargeted TIC7941PL_1, TIC7941PL_2, or TIC7941PL_3 or an untargetedTIC7941PL_1, TIC7941PL_2, or TIC7941PL_3. The codons of a recombinantnucleic acid molecule encoding for proteins disclosed herein can besubstituted by synonymous codons (known in the art as a silentsubstitution).

A recombinant DNA construct comprising a TIC7941 protein encodingsequence can further comprise a region of DNA that encodes for one ormore insect inhibitory agents which can be configured to concomitantlyexpress or co-express with a DNA sequence encoding a TIC7941 protein, aprotein different from a TIC7941 protein, an insect inhibitory dsRNAmolecule, or an ancillary protein. Ancillary proteins include, but arenot limited to, co-factors, enzymes, binding-partners, or other agentsthat function to aid in the effectiveness of an insect inhibitory agent,for example, by aiding its expression, influencing its stability inplants, optimizing free energy for oligomerization, augmenting itstoxicity, and increasing its spectrum of activity. An ancillary proteinmay facilitate the uptake of one or more insect inhibitory agents, forexample, or potentiate the toxic effects of the toxic agent.

A recombinant DNA construct can be assembled so that all proteins ordsRNA molecules are expressed from one promoter or each protein or dsRNAmolecules is under separate promoter control or some combinationthereof. The proteins of this invention can be expressed from amulti-gene expression system in which one or more proteins of theTIC7941 protein toxin class are expressed from a common nucleotidesegment which also contains other open reading frames and promoters,depending on the type of expression system selected. For example, abacterial multi-gene expression system can utilize a single promoter todrive expression of multiply-linked/tandem open reading frames fromwithin a single operon (i.e., polycistronic expression). In anotherexample, a plant multi-gene expression system can utilizemultiply-unlinked or linked expression cassettes, each cassetteexpressing a different protein or other agent such as one or more dsRNAmolecules.

Recombinant polynucleotides or recombinant DNA constructs comprising aTIC7941 protein encoding sequence can be delivered to host cells byvectors, e.g., a plasmid, baculovirus, synthetic chromosome, virion,cosmid, phagemid, phage, or viral vector. Such vectors can be used toachieve stable or transient expression of a TIC7941 protein encodingsequence in a host cell, or subsequent expression of the encodedpolypeptide. An exogenous recombinant polynucleotide or recombinant DNAconstruct that comprises a TIC7941 protein encoding sequence and that isintroduced into a host cell is referred in this application as a“transgene”.

Transgenic bacteria, transgenic plant cells, transgenic plants, andtransgenic plant parts that contain a recombinant polynucleotide thatexpresses TIC7941, TIC7941_His, TIC7941PL_1, TIC7941PL_2, or TIC7941PL_3protein encoding sequence is provided herein. The term “bacterial cell”or “bacterium” can include, but is not limited to, an Agrobacterium, aBacillus, an Escherichia, a Salmonella, a Pseudomonas, a Brevibacillus,a Klebsiella, an Erwinia, or a Rhizobium cell. The term “plant cell” or“plant” can include but is not limited to a dicotyledonous ormonocotyledonous plant. The term “plant cell” or “plant” can alsoinclude but is not limited to an alfalfa, banana, barley, bean,broccoli, cabbage, brassica, carrot, cassava, castor, cauliflower,celery, chickpea, Chinese cabbage, citrus, coconut, coffee, corn,clover, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus,flax, garlic, grape, hops, leek, lettuce, Loblolly pine, millets,melons, nut, oat, olive, onion, ornamental, palm, pasture grass, pea,peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine,radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum,Southern pine, soybean, spinach, squash, strawberry, sugar beet,sugarcane, sunflower, sweet corn, sweet gum, sweet potato, switchgrass,tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plantcell or plant. In certain embodiments, transgenic plants and transgenicplant parts regenerated from a transgenic plant cell are provided. Incertain embodiments, the transgenic plants can be obtained from atransgenic seed, by cutting, snapping, grinding or otherwisedisassociating the part from the plant. In certain embodiments, theplant part can be a seed, a boll, a leaf, a flower, a stem, a root, orany portion thereof, or a non-regenerable portion of a transgenic plantpart. As used in this context, a “non-regenerable” portion of atransgenic plant part is a portion that cannot be induced to form awhole plant or that cannot be induced to form a whole plant that iscapable of sexual and/or asexual reproduction. In certain embodiments, anon-regenerable portion of a plant part is a portion of a transgenicseed, boll, leaf, flower, stem, or root.

Methods of making transgenic plants that comprise insect,Lepidoptera-inhibitory amounts of a TIC7941 protein are provided. Suchplants can be made by introducing a recombinant polynucleotide thatencodes any of the proteins provided in this application into a plantcell, and selecting a plant derived from said plant cell that expressesan insect, Lepidoptera-inhibitory amount of the proteins. Plants can bederived from the plant cells by regeneration, seed, pollen, or meristemtransformation techniques. Methods for transforming plants are known inthe art.

Processed plant products, wherein the processed product comprises adetectable amount of a TIC7941 protein, an insect inhibitory segment orfragment thereof, or any distinguishing portion thereof, are alsodisclosed herein. In certain embodiments, the processed product isselected from the group consisting of plant parts, plant biomass, oil,meal, sugar, animal feed, flour, flakes, bran, lint, hulls, processedseed, and seed. In certain embodiments, the processed product isnon-regenerable. The plant product can comprise commodity or otherproducts of commerce derived from a transgenic plant or transgenic plantpart, where the commodity or other products can be tracked throughcommerce by detecting nucleotide segments or expressed RNA or proteinsthat encode or comprise distinguishing portions of a TIC7941 protein.

Plants expressing a TIC7941 protein can be crossed by breeding withtransgenic events expressing other toxin proteins and/or expressingother transgenic traits such as herbicide tolerance genes, genesconferring yield or stress tolerance traits, and the like, or suchtraits can be combined in a single vector so that the traits are alllinked.

As further described in the Examples, the TIC7941 protein toxin classand sequences having a substantial percentage identity to a member ofthe TIC7941 protein toxin class can be identified using methods known tothose of ordinary skill in the art such as polymerase chain reaction(PCR), thermal amplification and hybridization. For example, theproteins in the TIC7941 protein toxin class can be used to produceantibodies that bind specifically to related proteins, and can be usedto screen for and to find other protein members that are closelyrelated.

Furthermore, nucleotide sequences encoding the TIC7941 toxin proteinscan be used as probes and primers for screening to identify othermembers of the class using thermal-cycle or isothermal amplification andhybridization methods. For example, oligonucleotides derived fromsequences as set forth in SEQ ID NO:3, SEQ ID NO:11, and SEQ ID NO:13can be used to determine the presence or absence of a TIC7941 transgenein a deoxyribonucleic acid sample derived from a commodity product.Given the sensitivity of certain nucleic acid detection methods thatemploy oligonucleotides, it is anticipated that oligonucleotides derivedfrom sequences as set forth in SEQ ID NO:3, SEQ ID NO:11, and SEQ IDNO:13 can be used to detect a TIC7941PL_1, TIC7941PL_2, and TIC7941PL_3transgene in commodity products derived from pooled sources where only afraction of the commodity product is derived from a transgenic plantcontaining any of the transgenes. It is further recognized that sucholigonucleotides can be used to introduce nucleotide sequence variationin each of SEQ ID NO:3, SEQ ID NO:11, and SEQ ID NO:13. Such“mutagenesis” oligonucleotides are useful for identification of TIC7941protein toxin class amino acid sequence variants exhibiting a range ofinsect inhibitory activity or varied expression in transgenic plant hostcells.

Nucleotide sequence homologs, e.g., insecticidal proteins encoded bynucleotide sequences that hybridize to each or any of the sequencesdisclosed in this application under stringent hybridization conditions,are also an embodiment of the present invention. The invention alsoprovides a method for detecting a first nucleotide sequence thathybridizes to a second nucleotide sequence, wherein the first nucleotidesequence (or its reverse complement sequence) encodes a pesticidalprotein or pesticidal fragment thereof and hybridizes to the secondnucleotide sequence. In such case, the second nucleotide sequence can beany of the nucleotide sequences presented as SEQ ID NO:3, SEQ ID NO:1,SEQ ID NO: 11, and SEQ ID NO:13 under stringent hybridizationconditions. Nucleotide coding sequences hybridize to one another underappropriate hybridization conditions, such as stringent hybridizationconditions, and the proteins encoded by these nucleotide sequences crossreact with antiserum raised against any one of the other proteins.Stringent hybridization conditions, as defined herein, comprise at leasthybridization at 42° C. followed by two washes for five minutes each atroom temperature with 2×SSC, 0.1% SDS, followed by two washes for thirtyminutes each at 65° C. in 0.5×SSC, 0.1% SDS. Washes at even highertemperatures constitute even more stringent conditions, e.g.,hybridization conditions of 68° C., followed by washing at 68° C., in2×SSC containing 0.1% SDS.

One skilled in the art will recognize that, due to the redundancy of thegenetic code, many other sequences are capable of encoding such relatedproteins, and those sequences, to the extent that they function toexpress pesticidal proteins either in Bacillus strains or in plantcells, are embodiments of the present invention, recognizing of coursethat many such redundant coding sequences will not hybridize under theseconditions to the native Bacillus or Paenibacillus sequences encodingTIC7941. This application contemplates the use of these and otheridentification methods known to those of ordinary skill in the art toidentify TIC7941 protein-encoding sequences and sequences having asubstantial percentage identity to TIC7941 protein-encoding sequences.

This disclosure also contemplates the use of molecular methods known inthe art to engineer and clone commercially useful proteins comprisingchimeras of proteins from pesticidal proteins; e.g., the chimeras may beassembled from segments of the TIC7941-related proteins to deriveadditional useful embodiments including assembly of segments of TIC7941,TIC7941PL_1, TIC7941PL_2, and TIC7941PL_3 with segments of diverseproteins different from TIC7941, TIC7941PL_1, TIC7941PL_2, andTIC7941PL_3; and related proteins. The TIC7941 proteins may be subjectedto alignment to each other and to other Bacillus, Paenibacillus or otherpesticidal proteins (whether or not these are closely or distantlyrelated phylogenetically), and segments of each such protein may beidentified that are useful for substitution between the alignedproteins, resulting in the construction of chimeric proteins. Suchchimeric proteins can be subjected to pest bioassay analysis andcharacterized for the presence or absence of increased bioactivity orexpanded target pest spectrum compared to the parent proteins from whicheach such segment in the chimera was derived. The pesticidal activity ofthe polypeptides may be further engineered for activity to a particularpest or to a broader spectrum of pests by swapping domains or segmentswith other proteins or by using directed evolution methods known in theart.

In addition, this disclosure contemplates engineering a variantpesticidal protein by inserting peptide sequences within the nativepesticidal protein that can improve the pesticidal activity againstspecific insect pest species. The inserted peptide binds to an insectmidgut receptor. Specific binding of the endotoxin to specific receptorslocated in the insect midgut is one step in the mode of pesticidalaction of a pesticidal protein. At least five different proteinreceptors have been described to be involved in interactions leading toinsect mortality: a cadherin-like protein (CADR), aglycosylphosphatidyl-inositol (GPI)-anchored aminopeptidase-N (APN), aGPI-anchored alkaline phosphatase (ALP), a transmembrane ABCtransporter, and an “A Disentegrin And Metalloprotease” or ADAMmetalloprotease. In addition, it has been proposed that glycolipids arealso important Cry-receptor molecules in insects and nematodes (Pigottet al. (2007) Role of Receptors in Bacillus thuringiensis Crystal ToxinActivity. Microbiology and Molecular Biology Reviews, 71(2): 255-281;Ochoa-Campuzano et al. (2007) An ADAM metalloprotease is a Cry3AaBacillus thuringiensis toxin receptor. Biochemical and BiophysicalResearch Communication, 362(2): 437-442). The peptide fragment,FAWPEPBIN binds to the Fall Armyworm (FAW) transmembrane ABC transporterABCc4. Insertion of the coding sequence, FAWPEPBIN_Bac (SEQ ID NO:15),encoding the peptide FAWPEPBIN (SEQ ID NO:17) within the domain 2 loopof TIC7941 increased pesticidal activity against FAW in certainvariants. Specifically, insertion of FAWPEPBIN in amino acid positions805-816 in TIC7941_2His resulted in little or no demonstrated activityagainst FAW whereas insertion of FAWPEPBIN in amino acid positions804-815 of TIC7941_3His demonstrated activity against FAW.

A synthetic DNA sequence encoding the FAWPEPBIN peptide, FAWPEPBIN_PL,(SEQ ID NO:16) was designed for expression in a plant cell. FAWPEPBIN_PLis found between nucleotide positions 2386 and 2421 of the syntheticcoding sequence TIC7941PL_2 and within nucleotide positions 2383-2418 ofthe TIC7941PL_3 synthetic coding sequence. The FAWPEPBIN peptidefragment is located within amino acid positions 796-807 of TIC7941PL_2and 795-806 of TIC7941PL_3. Corn plants were transformed with binaryvectors comprising transgene cassettes used for the expression ofTIC7941PL_2 and TIC7941PL_3. The plants expressing TIC7941PL_2 andTIC7941PL_3 will be used to assay the pesticidal activity of theengineered toxins against FAW.

Methods of controlling insects, in particular Lepidoptera infestationsof crop plants, with the TIC7941 proteins are also disclosed in thisapplication. Such methods can comprise growing a plant comprising aninsect- or Lepidoptera-inhibitory amount of a TIC7941 toxin protein. Incertain embodiments, such methods can further comprise any one or moreof: (i) applying any composition comprising or encoding a TIC7941 toxinprotein to a plant or a seed that gives rise to a plant; and (ii)transforming a plant or a plant cell that gives rise to a plant with apolynucleotide encoding a TIC7941 toxin protein. In general, it iscontemplated that a TIC7941 toxin protein can be provided in acomposition, provided in a microorganism, or provided in a transgenicplant to confer insect inhibitory activity against Lepidopteran insects.

In certain embodiments, a recombinant nucleic acid molecule of a TIC7941toxin protein is the pesticidally active ingredient of an insectinhibitory composition prepared by culturing recombinant Bacillus or anyother recombinant bacterial cell transformed to express a TIC7941 toxinprotein under conditions suitable to express the TIC7941 toxin protein.Such a composition can be prepared by desiccation, lyophilization,homogenization, extraction, filtration, centrifugation, sedimentation,or concentration of a culture of such recombinant cellsexpressing/producing said recombinant polypeptide. Such a process canresult in a Bacillus or other entomopathogenic bacterial cell extract,cell suspension, cell homogenate, cell lysate, cell supernatant, cellfiltrate, or cell pellet. By obtaining the recombinant polypeptides soproduced, a composition that includes the recombinant polypeptides caninclude bacterial cells, bacterial spores, and parasporal inclusionbodies and can be formulated for various uses, including as agriculturalinsect inhibitory spray products or as insect inhibitory formulations indiet bioassays.

In one embodiment, to reduce the likelihood of resistance development,an insect inhibitory composition comprising a TIC7941 toxin protein canfurther comprise at least one additional polypeptide that exhibitsinsect inhibitory activity against the same Lepidopteran insect species,but which is different from the TIC7941 toxin protein. Possibleadditional polypeptides for such a composition include any insectinhibitory protein or insect inhibitory dsRNA molecule known to a personof ordinary skill in the art. One example for the use of suchribonucleotide sequences to control insect pests is described in Baum,et al. (U.S. Patent Publication 2006/0021087 A1). Such additionalpolypeptide for the control of Lepidopteran pests may be selected fromthe group consisting of an insect inhibitory protein, such as, but notlimited to, Cry1A (U.S. Pat. No. 5,880,275), Cry1Ab, Cry1Ac, Cry1A.105,Cry1Ae, Cry1B (U.S. Patent Publication No. 10/525,318), Cry1C (U.S. Pat.No. 6,033,874), Cry1D, Cry1Da and variants thereof, Cry1E, Cry1F, andCry1A/F chimeras (U.S. Pat. Nos. 7,070,982; 6,962,705; and 6,713,063),Cry1G, Cry1H, Cry1 I, Cry1J, Cry1K, Cry1L, Cry1-type chimeras such as,but not limited to, TIC836, TIC860, TIC867, TIC869, and TIC1100(International Application Publication WO2016/061391 (A2)), TIC2160(International Application Publication WO2016/061392(A2)), Cry2A, Cry2Ab(U.S. Pat. No. 7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15,Cry43A, Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A,VIP3Ab, VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AND AXMI-045(U.S. Patent Publication 2013-0117884 A1), AXMI-52, AXMI-58, AXMI-88,AXMI-97, AXMI-102, AXMI-112, AXMI-117, AXMI-100 (U.S. Patent Publication2013-0310543 A1), AXMI-115, AXMI-113, AXMI-005 (U.S. Patent Publication2013-0104259 A1), AXMI-134 (U.S. Patent Publication 2013-0167264 A1),AXMI-150 (U.S. Patent Publication 2010-0160231 A1), AXMI-184 (U.S.Patent Publication 2010-0004176 A1), AXMI-196, AXMI-204, AXMI-207,AXMI-209 (U.S. Patent Publication 2011-0030096 A1), AXMI-218, AXMI-220(U.S. Patent Publication 2014-0245491 A1), AXMI-221z, AXMI-222z,AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication 2014-0196175A1), AXMI-238 (U.S. Patent Publication 2014-0033363 A1), AXMI-270 (U.S.Patent Publication 2014-0223598 A1), AXMI-345 (U.S. Patent Publication2014-0373195 A1), AXMI-335 (International Application PublicationWO2013/134523(A2)), DIG-3 (U.S. Patent Publication 2013-0219570 A1),DIG-5 (U.S. Patent Publication 2010-0317569 A1), DIG-11 (U.S. PatentPublication 2010-0319093 A1), AfIP-1A and derivatives thereof (U.S.Patent Publication 2014-0033361 A1), AfIP-1B and derivatives thereof(U.S. Patent Publication 2014-0033361 A1), PIP-1APIP-1B (U.S. PatentPublication 2014-0007292 A1), PSEEN3174 (U.S. Patent Publication2014-0007292 A1), AECFG-592740 (U.S. Patent Publication 2014-0007292A1), Pput_1063 (U.S. Patent Publication 2014-0007292 A1), DIG-657(International Application Publication WO2015/195594 A2), Pput_1064(U.S. Patent Publication 2014-0007292 A1), GS-135 and derivativesthereof (U.S. Patent Publication 2012-0233726 A1), GS153 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS154 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS155 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2012-0167259A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2012-0047606 A1, SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Patent Publication 2011-0154536 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2011-0112013A1, SEQ ID NO:2 and 4 and derivatives thereof as described in U.S.Patent Publication 2010-0192256 A1, SEQ ID NO:2 and derivatives thereofas described in U.S. Patent Publication 2010-0077507 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2010-0077508A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2009-0313721 A1, SEQ ID NO:2 or 4 and derivatives thereof asdescribed in U.S. Patent Publication 2010-0269221 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Pat. No. 7,772,465 (B2),CF161_0085 and derivatives thereof as described in WO2014/008054 A2,Lepidopteran toxic proteins and their derivatives as described in USPatent Publications US2008-0172762 A1, US2011-0055968 A1, andUS2012-0117690 A1; SEQ ID NO:2 and derivatives thereof as described inU.S. Pat. No. 7,510,878(B2), SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Pat. No. 7,812,129(B1), DIG-911 and DIG-180 asdescribed in US Patent Publication No. 2015-0264940A1; and the like.

In other embodiments, such composition/formulation can further compriseat least one additional polypeptide that exhibits insect inhibitoryactivity to an insect that is not inhibited by an otherwise insectinhibitory protein of the present invention to expand the spectrum ofinsect inhibition obtained. For example, for the control of Hemipteranpests, combinations of insect inhibitory proteins of the presentinvention can be used with Hemipteran-active proteins such as TIC1415(US Patent Publication 2013-0097735 A1), TIC807 (U.S. Pat. No.8,609,936), TIC834 (U.S. Patent Publication 2013-0269060 A1), AXMI-036(U.S. Patent Publication 2010-0137216 A1), and AXMI-171 (U.S. PatentPublication 2013-0055469 A1). Further a polypeptide for the control ofColeopteran pests may be selected from the group consisting of an insectinhibitory protein, such as, but not limited to, Cry3Bb (U.S. Pat. No.6,501,009), Cry1C variants, Cry3A variants, Cry3, Cry3B, Cry34/35, 5307,AXMI134 (U.S. Patent Publication 2013-0167264 A1) AXMI-184 (U.S. PatentPublication 2010-0004176 A1), AXMI-205 (U.S. Patent Publication2014-0298538 A1), AXMI-207 (U.S. Patent Publication 2013-0303440 A1),AXMI-218, AXMI-220 (U.S. Patent Publication 20140245491A1), AXMI-221z,AXMI-223z (U.S. Patent Publication 2014-0196175 A1), AXMI-279 (U.S.Patent Publication 2014-0223599 A1), AXMI-R1 and variants thereof (U.S.Patent Publication 2010-0197592 A1, TIC407, TIC417, TIC431, TIC807,TIC853, TIC901, TIC1201, TIC3131, DIG-10 (U.S. Patent Publication2010-0319092 A1), eHIPs (U.S. Patent Application Publication No.2010/0017914), IP3 and variants thereof (U.S. Patent Publication2012-0210462 A1), PHI-4 variants (U.S. Patent Application Publication2016-0281105 A1), PIP-72 variants (WO 2016-144688 A1), PIP-45 variants,PIP-64 variants, PIP-74 variants, PIP-75 variants, and PIP-77 variants(WO 2016-144686 A1), DIG-305 (WO 2016109214 A1), PIP-47 variants (U.S.Patent Publication 2016-0186204 A1), DIG-17, DIG-90, DIG-79 (WO2016-057123 A1), DIG-303 (WO 2016-070079 A1), and W-Hexatoxin-Hv1a (U.S.Patent Application Publication US2014-0366227 A1).

Additional polypeptides for the control of Coleopteran, Lepidopteran,and Hemipteran insect pests can be found on the Bacillus thuringiensistoxin nomenclature website maintained by Neil Crickmore (accessible onthe internet at www.btnomenclature.info).

The possibility for insects to develop resistance to certaininsecticides has been documented in the art. One insect resistancemanagement strategy is to employ transgenic crops that express twodistinct insect inhibitory agents that operate through different modesof action. Therefore, any insects with resistance to either one of theinsect inhibitory agents can be controlled by the other insectinhibitory agent. Another insect resistance management strategy employsthe use of plants that are not protected to the targeted Lepidopteranpest species to provide a refuge for such unprotected plants. Oneparticular example is described in U.S. Pat. No. 6,551,962, which isincorporated by reference in its entirety.

Other embodiments such as topically applied pesticidal chemistries thatare designed for controlling pests that are also controlled by theproteins disclosed herein to be used with proteins in seed treatments,spray on, drip on, or wipe on formulations can be applied directly tothe soil (a soil drench), applied to growing plants expressing theproteins disclosed herein, or formulated to be applied to seedcontaining one or more transgenes encoding one or more of the proteinsdisclosed. Such formulations for use in seed treatments can be appliedwith various stickers and tackifiers known in the art. Such formulationscan contain pesticides that are synergistic in mode of action with theproteins disclosed, so that the formulation pesticides act through adifferent mode of action to control the same or similar pests that canbe controlled by the proteins disclosed, or that such pesticides act tocontrol pests within a broader host range or plant pest species that arenot effectively controlled by the TIC7941 pesticidal proteins.

The aforementioned composition/formulation can further comprise anagriculturally-acceptable carrier, such as a bait, a powder, dust,pellet, granule, spray, emulsion, a colloidal suspension, an aqueoussolution, a Bacillus spore/crystal preparation, a seed treatment, arecombinant plant cell/plant tissue/seed/plant transformed to expressone or more of the proteins, or bacterium transformed to express one ormore of the proteins. Depending on the level of insect inhibitory orpesticidal inhibition inherent in the recombinant polypeptide and thelevel of formulation to be applied to a plant or diet assay, thecomposition/formulation can include various by weight amounts of therecombinant polypeptide, e.g. from 0.0001% to 0.001% to 0.01% to 1% to99% by weight of the recombinant polypeptide.

This disclosure also contemplates compositions and methods for reducingexpression of a pesticidal protein in the reproductive tissues of atransgenic plant through the use of microRNAs (miRNAs). miRNAs areessential components of the gene silencing machinery in plants. Inplants, the production of miRNAs is a tissue-specific process, istightly associated with transcription and splicing, and even variesbetween miRNA precursors. Encoded by nuclear DNA in plants, miRNAsfunction via base-pairing with complementary sequences within mRNAmolecules (Achkar et al. (2016) miRNA Biogenesis: A Dynamic Pathway,Trends in Plant Science. 21(12): 1034-1044). miRNAs are produced from aprimary miRNA transcript (pri-miRNA). The nascent pri-miRNAs are cappedat the 5′ end and polyadenylated at the 3′ end, and intron-containingpri-miRNAs are spliced or alternatively spliced. pri-miRNAs areprocessed by the dicing complex which contains the nuclear RNaseDICER-LIKE 1 (DCL1) and its accessory proteins SERRATE (SE) andHYPONASTIC LEAVES (HYL1) as core components, to yield mature twenty-one(21) nucleotide miRNA/miRNA® duplexes. The miRNA/miRNA® duplex isstabilized through 3′-terminal 2′-O-methylation by HUA ENAHANCER 1(HENI). HENI also contributes to export of the miRNA/miRNA® duplex fromthe cell nucleus and RNA-induced silencing complex (RISC) assembly.During RICS loading, one strand of the small RNA duplex is selected asthe guide strand and incorporated into ARGONAUTE 1 (AGO1) to form afunctional RISC, whereas the other strand (the passenger strand) isremoved and degraded. The loading of miRNAs into AGO proteins isaffected by the bulges in the miRNA/miRNA® duplexes caused by base pairmismatches. AGO1 prefers duplexes with central mismatches (Yu et al.(2017) The “how” and “where” of plant microRNAs. New Phytologist, 216:1002-1017).

Plant miRNAs regulate target genes at the post-transcriptional level viatwo major mechanisms: transcript cleavage and translation repression. Inplants, translation repression is less frequently observed thantranscript cleavage. miRNA-guided RNA cleavage occurs at a preciseposition in the target mRNA. Cleavage is accomplished by the PIWI domainof AGO proteins, which forms an RNase H-like fold and exhibitsendonuclease activity. The 5′ and 3′ cleavage fragments are subsequentlydegraded by exonucleases. Known factors required for miRNA-mediatedtranslation inhibition include the microtubule-severing enzyme KATANIN 1(KTN1), the processing body (P body) component of VARICOSE (VCS), theGW-repeat protein SUO, and the ER membrane protein ALTERED MERISTEMPROGRAM 1 (AMP1). Mutations in these genes selectively interfere withmiRNA-guided repression at the protein level, suggesting that transcriptcleavage and translation repression are two independent modes of action.The molecular mechanism underlying miRNA-mediated translation repressionis not well understood. In vitro analysis suggests that plant miRNAscould inhibit translation initiation or hinder the movement of ribosomes(Yu et al. (2017) The “how” and “where” of plant microRNAs. NewPhytologist, 216: 1002-1017).

In addition to mRNA cleavage and translation repression, some miRNAsalso trigger the production of secondary short interfering RNAs (siRNAs)from their transcripts, and this is a widespread and conservedphenomenon in plants (Yu et al. (2017) The “how” and “where” of plantmicroRNAs. New Phytologist, 216: 1002-1017). The miRNAs that typicallytrigger the production of these secondary siRNAs are twenty-two (22)nucleotides in length as opposed to the twenty-one (21) nucleotidemiRNAs described above. The targeted RNA is converted intodouble-stranded RNA (dsRNA) by RNA-dependent RNA polymerase (RdRp),which is then cleaved into siRNAs by DCL nucleases. Typically, onestrand of the duplex preferentially associates with an AGO protein toform an effector complex (RNA-induced silencing complex, or RISC), thattargets and silences transcripts based on sequence complementarity. InArabidopsis, after AGO1-mediated miRNA-guided RNA cleavage of the targetRNA, either the 5′ or 3′ cleavage fragment is stabilized by SUPPRESSOROF GENE SILENCING 3 (SGS3), which associates with RISC by recognizingfeatures of the twenty-two (22) nucleotide miRNA/target duplex toprotect the cleavage. RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) is recruitedto convert the cleavage fragment into dsRNA which is later diced intosiRNAs at a twenty-one (21) nucleotide interval fragment fromdegradation. In plants, this process can be amplified through productionof secondary siRNAs after transcription by RNA-dependent RNA polymerase(RdRp) on the primary target RNA. (Cuperus et al., (2010) UniqueFunctionality of 22 nt miRNAs in Triggering RDR6-Dependent siRNABiogenesis from Target Transcripts in Arabidopsis. Nat Struct Mol Biol,17(8): 997-1003; Chen et al., (2010) 22-Nucleotide RNAs triggersecondary siRNA biogenesis in Plants. Proceedings of the NationalAcademy of Sciences, 107: 15269-15274; Yu et al. (2017) The “how” and“where” of plant microRNAs. New Phytologist, 216: 1002-1017).

Through data mining of miRNAs in various tissues in soybean, two miRNAswere identified that were over-represented in reproductive tissues whencompared to vegetative tissues; miR395 and miR4392. miR395 is processedinto a twenty-one (21) nucleotide miRNA/miRNA® duplex and is expressedmostly in the soybean flower stamen. miR4392 is processed into atwenty-two (22) nucleotide miRNA/miRNA® duplex and triggers theproduction of secondary siRNAs from its transcripts, amplifying thesuppression signal. miR4392 is highly enriched in the soybean floweranthers. Bound with an ARGO protein to form a silencing complex, miRNAsfunction as sequence-specific guides, directing the silencing complex totranscripts through base pairing between the miRNA and complementarysites herein referred to as “miRNA target binding sites”, within the 3′untranslated region (3′ UTR) of the target RNAs. miRNA target bindingsites corresponding to miR395 (Gm.miR395_1 (SEQ ID NO:18) andGm.miR395_2 (SEQ ID NO:19)) and miR4392 (Gm.miR4392_1 (SEQ ID NO:21) andGm.miR4392_2 (SEQ ID NO:22)) were operably linked using a DNA spacer(SP-ART.8a-1, SEQ ID NO:24) to construct SUP-miR395 (SEQ ID NO:20) andSUP-miR4392 (SEQ ID NO:23), respectively. SUP-miR395 and SUP-miR4392were in turn operably linked to the TIC7941PL_1 coding sequence 3′ afterthe stop codon producing the transgenes, TIC7941PL_1-miR395 (SEQ IDNO:25) and TIC7941PL_1-miR4392 (SEQ ID NO:26), respectively. Expressionof TIC7941PL_1-miR395 and TIC7941PL_1-miR4392 had no effect on thepesticidal activity of TIC7941PL_1. Both TIC7941PL_1-miR395 andTIC7941PL_1-miR4392 demonstrated similar pesticidal activity againstSouthern armyworm (SAW, Spodoptera eridania), Soybean looper (SBL,Chrysodeixis includens), and Soybean podworm (SPW, Helicoverpa zea) whencompared to TIC7941PL_1 in leaf disc assay. Operably linking the miR395and miR4392 target sites to the TIC7941PL_1 coding sequence is intendedto lower expression of the TIC7941PL_1 pesticidal protein in thereproductive tissues of transgenic soybean expressing TIC7941PL_1-miR395or TIC7941PL_1-miR4392.

In view of the foregoing, those of skill in the art should appreciatethat changes can be made in the specific aspects which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention. Thus, specific structural and functionaldetails disclosed herein are not to be interpreted as limiting. Itshould be understood that the entire disclosure of each reference citedherein is incorporated within the disclosure of this application.

EXAMPLES Example 1 Discovery, Cloning, and Expression of TIC7941

A sequence encoding a novel Paenibacillus lentimorbus pesticidal proteinwas identified, cloned, sequence confirmed, and tested in insectbioassay. The pesticidal protein, TIC7941 isolated from thePaenibacillus lentimorbus species DSC020651, represents a novelVip3C-like protein. Distant-related sequences to TIC7941 are Vip3Cal (at72.43% identity, the closest known relative), Vip3Aa1 (64.45% identity),and a Vip3B-like protein (59% identity).

A full length copy of the coding region for TIC7941 and a His-taggedversion of TIC7941 (TIC7941_His) were synthesized by methods known inthe art and comprise the translational initiation and termination codonsof each coding sequence. The TIC7941 coding sequence was cloned usingmethods known in the art into a Bt expression vector in operable linkagewith a Bt expressible promoter. The Bt expression vector comprised apromoter that is on during the sporulation stage of the bacillus. Inaddition, the TIC7941_His coding sequence was cloned into a vector usedfor protein expression in Escherichia coli (E. coli). For isolation ofthe E. coli expressed proteins, a Histidine tag was operably linked tothe expressed coding sequences to facilitate column purification of theprotein. The coding sequences and their respective protein sequencesused for bacterial expression are presented in Table 2.

TABLE 2 Toxin coding sequences and corresponding protein sequences usedfor expression in Bt and E. coli. DNA Coding Bacterial Sequence ProteinExpression Toxin SEQ ID NO: SEQ ID NO: Host TIC7941 1 2 Bt TIC7941_His 56 E. coli

Example 2 TIC7941 Demonstrates Lepidopteran Activity in Insect Bioassay

The pesticidal protein TIC7941 was expressed in Bt and E. coli andassayed for toxicity to various species of Lepidoptera, Coleoptera,Hemiptera, and Dipteran. Preparations of TIC7941 and TIC7941_His fromboth Bt and E. coli, were assayed against the Lepidopteran species Blackcutworm (BCW, Agrotis ipsilon), Corn earworm (CEW, also known as Soybeanpodworm (SPW), Helicoverpa zea), European corn borer (ECB, Ostrinianubilalis), Fall armyworm (FAW, Spodoptera frugiperda), Southernarmyworm (SAW, Spodoptera eridania), Soybean looper (SBL, Chrysodeixisincludens), Southwestern corn borer (SWCB, Diatraea grandiosella),Tobacco budworm (TBW, Heliothis virescens), and Velvet bean caterpillar(VBW, Anticarsia gemmatalis); the Coleopteran species Colorado potatobeetle (CPB, Leptinotarsa decemlineata), and Western Corn Rootworm (WCB,Diabrotica virgifera virgifera); and the Hemipteran species Tarnishedplant bug (TPB, Lygus lineolaris) and Western tarnished plant bug (WTP,Lygus hesperus); and the Dipteran species Yellow Fever Mosquito (YFM,Aedes aegypti).

To produce TIC7941 in Bt hosts, a Bt strain expressing TIC7941 was grownfor twenty four (24) hours and then the culture was added to insectdiet. Mortality and stunting were evaluated by comparing the growth anddevelopment of insects on a diet with a culture from the Bt strainexpressing TIC7941 to insects on a diet with an untreated controlculture.

The E. coli strain expressing TIC7941_His was treated in a similarmanner to the Bt strain and was provided in an insect diet after proteinpurification and compared to the growth and development of insects on adiet with an untreated control culture. TIC7941 demonstrated pesticidalactivity against the Lepidopteran insect pest species Black cutworm,Corn earworm, European corn borer, Southern armyworm, Soybean looper andSouthwestern corn borer. Activity was particularly high against Soybeanlooper.

Example 3 Assay of TIC7941PL_1 Activity Against Lepidopteran Pests inStably Transformed Corn Plants

A binary plant transformation vector comprising a transgene cassettedesigned to express untargeted TIC7941PL_1 pesticidal protein was clonedusing methods known in the art. The resulting vector was used to stablytransform corn plants. Tissues were harvested from the transformants andused in insect bioassay against various Lepidopteran insect pests.

A synthetic coding sequence was constructed for use in expression of theTIC7941 in plants, cloned into a binary plant transformation vector, andused to transform corn plant cells. The synthetic sequence wassynthesized, according to methods generally described in U.S. Pat. No.5,500,365, to avoid certain inimical problem sequences such as ATTA andA/T rich plant polyadenylation sequences while preserving the amino acidsequence of the native Paenibacillus protein. The synthetic codingsequence (SEQ ID NO:3) encodes a TIC7941PL_1 protein (SEQ ID NO:4) whichcomprises an additional alanine residue immediately following theinitiating methionine relative to the TIC7941 protein. The resultingplant transformation vector comprised a first transgene cassette forexpression of the TIC7941PL_1 pesticidal protein which comprised aconstitutive promoter, operably linked 5′ to a leader, operably linked5′ to an intron, operably linked 5′ to the synthetic coding sequenceencoding an untargeted TIC7941PL_1 protein (SEQ ID NO:4), which was inturn operably linked 5′ to a 3′ UTR; and a second transgene cassette forthe selection of transformed plant cells using glyphosate selection.

Corn plant cells were transformed with the binary transformation vectoras described above using an Agrobacterium-mediated transformationmethod. The transformed cells were induced to form plants by methodsknown in the art. Bioassays using plant leaf disks were performedanalogous to those described in U.S. Pat. No. 8,344,207. A singlefreshly hatched neonate larvae less than one day old was placed on eachleaf disc sample and allowed to feed for approximately four days. Anon-transformed corn plant was used to obtain tissue to be used as anegative control. Multiple transformation R₀ single-copy insertionevents from each binary vector were assessed against BCW, CEW, FAW, andSWCB.

Twelve transformed R₀ events were evaluated using plant leaf discs. Aleaf damage ratings (LDR) of one, three, or four was given for eachevent for each insect pest species assayed. An LDR of one (1) isequivalent to less than or equal to thirty percent damage. An LDR ofthree (3) is equivalent to thirty percent to less than or equal to fiftypercent damage. An LDR of four (4) is equivalent to greater than fiftypercent damage. The LDR scores for each event and each insect pestspecies is presented in Table 3.

TABLE 3 Leaf damage ratings (LDR) for transformed corn R₀ eventsexpressing TIC7941PL_1. R₀ Leaf Damage Ratings Event BCW CEW FAW SWCBEvent 1 1 1 4 3 Event 2 1 1 4 1 Event 3 4 4 4 4 Event 4 4 4 4 4 Event 51 1 4 3 Event 6 1 1 4 1 Event 7 4 4 4 4 Event 8 4 4 4 4 Event 9 1 1 4 4Event 10 4 4 4 4 Event 11 1 1 4 3 Event 12 1 1 4 1

As can be seen in Table 3, seven out of the twelve transformed R₀ eventsassayed demonstrated resistance to BCW and CEW. Three of the sevenevents also demonstrated resistance to SWCB.

Events one through six were selected for assay at the F₁ generation.Table 4 shows the LDR scores for each of the six events assayed againstthe four insect pest species.

TABLE 4 Leaf damage ratings (LDR) for transformed corn F₁ eventsexpressing TIC7941PL_1. F₁ Leaf Damage Ratings Event BCW CEW FAW SWCBEvent 1 1 1 4 1 Event 2 1 3 4 1 Event 3 1 1 4 3 Event 4 1 1 4 3 Event 51 1 4 3 Event 6 1 1 4 3

As can be seen in Table 4, all six events demonstrated resistanceagainst BCW, five of the six events demonstrated resistance against CEW,and two of the six events demonstrated resistance against SWCB. Cornplants stably transformed with a transgene cassette for the expressionof TIC7941 demonstrates resistance to Lepidopteran pest species such asBCW, CEW, and SWCB.

Example 4 Assay of TIC7941PL_1 Activity Against Lepidopteran Pests inStably Transformed Soybean Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express untargeted TIC7941PL_1 pesticidal protein werecloned using methods known in the art. The resulting vectors were usedto stably transform soybean plants. Tissues were harvested from thetransformants and used in insect bioassay against various Lepidopteraninsect pests.

The synthetic TIC7941PL_1 coding sequence designed for plant expressionas described in Example 3 was cloned into binary plant transformationvectors, and used to transform soybean plant cells. The binary vectorscomprising an untargeted TIC7941PL_1 coding sequence were constructedusing methods known in the art. The resulting plant transformationvectors comprised a first transgene cassette for expression of theTIC7941PL_1 pesticidal protein which comprised a plant expressiblepromoter, operably linked 5′ to a leader, operably linked 5′ to asynthetic coding sequence encoding an untargeted TIC7941PL_1 protein(SEQ ID NO:4), which was in turn operably linked 5′ to a 3′ UTR and; asecond transgene cassette for the selection of transformed plant cellsusing spectinomycin selection. Four (4) binary transformation vectorswere constructed as described above. Each construct comprised aTIC7941PL_1 expression cassette comprising different promoters and 3′UTRs.

The transformed soybean cells were induced to form plants by methodsknown in the art. Bioassays using plant leaf disks were performedanalogous to those described in U.S. Pat. No. 8,344,207. Anon-transformed soybean plant was used to obtain tissue to be used as anegative control. Multiple transformation events from each binary vectorwere assessed against SAW, SBL, SPW, and VBW.

R₀ events, derived from transformations using the four different binaryconstructs, were evaluated using plant leaf discs. A leaf damage rating(LDR) of one through four was given for each event for each insect pestspecies assayed. An LDR of one (1) is equivalent to less than or equalto twenty percent damage. An LDR of two (2) is equivalent to twentypercent to less than or equal to thirty five percent damage. An LDR ofthree (3) is equivalent to thirty five percent to less than or equal toseventy percent damage. An LDR of four (4) is equivalent to greater thanseventy percent damage. The LDR scores for each construct and eachinsect pest species is presented in Table 5. The number of eventsdemonstrating the LDR score (observed) relative to the number of eventsassayed is also provided. High penetrance of the resistance trait isdefined as an LDR score of one (1) wherein greater than fifty percent(50%) of the events demonstrate an LDR of one (1).

TABLE 5 Leaf damage ratings (LDR) and penetrance for transformed soybeanR₀ events expressing TIC7941PL_1. LDR (Observed/Assayed) Construct SAWSBL SPW VBC Construct 1 1 (12/14) 1 (13/14) 1 (12/14) 2 (1/13) Construct2 1 (14/14) 1 (14/14) 1 (13/14) 3 (9/14) Construct 3 1 (12/12) 1 (12/12)1 (12/12) 3 (5/12) Construct 4 1 (12/15) 1 (15/15) 1 (12/15)  3 (10/15)

As can be seen in Table 5, R₀ soybean events expressing TIC7941PL_1transformed with each of the four (4) constructs demonstrated highresistance with high penetrance to SAW, SBL, and SPW. Stably transformedsoybean plants expressing TIC7941PL_1 demonstrate resistance toLepidopteran pest species, and is highly efficacious against SAW, SBL,and SPW.

Example 5 Assay of TIC7941PL_1 Activity Against Lepidopteran Pests inStably Transformed Cotton Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express both plastid targeted and untargeted TIC7941PL_1pesticidal protein are cloned using methods known in the art. Theresulting vectors are used to stably transform cotton plants. Tissuesare harvested from the transformants and used in insect bioassay againstvarious Lepidopteran insect pests.

The synthetic coding sequence designed for plant expression as describedin Example 3 is cloned into binary plant transformation vectors, andused to transform cotton plant cells. Binary vectors comprising plastidtargeted and untargeted TIC7941PL_1 coding sequences are constructedusing methods known in the art. The resulting plant transformationvectors comprise a first transgene cassette for expression of theTIC7941PL_1 pesticidal protein which comprises a constitutive promoter,operably linked 5′ to a leader, operably linked 5′ to a synthetic codingsequence encoding a plastid targeted or untargeted TIC7941PL_1 protein,which is in turn operably linked 5′ to a 3′ UTR and; a second transgenecassette for the selection of transformed plant cells usingspectinomycin selection.

The transformed cotton cells are induced to form plants by methods knownin the art. Bioassays using plant leaf disks are performed analogous tothose described in U.S. Pat. No. 8,344,207. A non-transformed cottonplant is used to obtain tissue to be used as a negative control.Multiple transformation events from each binary vector are assessedagainst CBW, FAW, SBL, and TBW, as well as any other Lepidopteran insectpest species known to cause agronomic damage to cotton crops.

In addition to leaf discs, other tissues can also be used to assessresistance imparted by expression of TIC7941PL_1 toxin protein intransgenic cotton plants, such as squares and bolls. Damage ratingscores are applied to each sample corresponding to each insect pest andcompared to negative controls to determine if expression of TIC7941PL_1provides resistance to a particular insect pest species.

Example 6 Improving the Pesticidal Activity of TIC7941 Against FallArmyworm

This example illustrates the improvement of the pesticidal activity ofTIC7941 against Fall armyworm through insertion of a FAW transmembraneABC transporter (ABCc4) binding peptide into the TIC7941 proteinsequence.

The peptide fragment FAWPEPBIN (presented as SEQ ID NO:17) binds to theFAW transmembrane ABC transporter ABCc4. FAWPEPBIN_Bac (SEQ ID NO:15) isa synthetic coding sequence encoding FAWPEPBIN (SEQ ID NO:17) forexpression of the FAWPEPBIN peptide in bacteria.

Engineered His-tagged TIC7941 proteins with the FAWPEPBIN peptideinserted into different positions in the domain 2 loop of the proteinwere compared in insect bioassay. The TIC7941_2His coding sequence (SEQID NO:7) encodes the TIC7941_2His pesticidal protein (SEQ ID NO:8). TheTIC7941_3His coding sequence (SEQ ID NO:9) encodes the TIC7941_3Hispesticidal protein (SEQ ID NO:10). The FAWPEPBIN_Bac synthetic codingsequence is found within nucleotide positions 2413-2448 of TIC7941_2Hisand within positions 2410-2445 of TIC7941_3His. The FAWPEPBIN peptidesequence is located at amino acid positions 805 to 816 of TIC7941_2Hisand amino acid positions 804 to 815 of TIC7941_3His.

The pesticidal activity of the TIC7941_His, TIC7941_2His, andTIC7941_3His pesticidal proteins were assayed against FAW. BothTIC7941_His and TIC7941_2His demonstrated little or no activity againstFAW. However, TIC7941_3His demonstrated improved pesticidal activityagainst FAW. Thus, insertion of the synthetic coding sequenceFAWPEPBIN_Bac in the amino acid positions 804-815 of TIC7941_3Hisimproved the pesticidal activity of the TIC7941 protein against FAW.

Example 7 Assay of Activity of TIC7941PL_2 and TIC7941PL_3 Against FallArmyworm in Stably Transformed Corn Plants

Binary plant transformation vectors comprising transgene cassettesdesigned to express the TIC7941PL_2 and TIC7941PL_3 pesticidal proteinsare cloned using methods known in the art. The resulting vectors areused to stably transform corn plants. Tissues are harvested from thetransformants and used in insect bioassay against FAW and otherLepidopteran insect pests.

Binary plant transformation vectors are constructed as previouslydescribed in Example 3. The binary vectors comprise a transgene cassetteused to express TIC7941PL_2 or TIC7941PL_3. TIC7941PL_2 and TIC7941PL_3comprise the ABCc4 receptor binding peptide FAWPEPBIN. A synthetic DNAsequence (FAWPEPBIN_PL, SEQ ID NO:16) used for expression in a plantcell and encoding the Fall armyworm transmembrane ABC transporter ABCc4binding peptide FAWPEPBIN, is inserted into the TIC7941PL_1 toxinprotein. The FAWPEPBIN_PL encoding DNA fragment is found withinnucleotide positions 2386-2421 of TIC7941PL_2 and within 2383-2418 ofTIC7941PL_3. The FAWPEPBIN peptide fragment is located at amino acidpositions 796-807 of TIC7941PL_2 and 795-806 of TIC7941PL_3.

Corn plant cells are transformed with the binary transformation vectorsas described above using an Agrobacterium-mediated transformationmethod. The transformed cells are induced to form plants by methodsknown in the art. Bioassays using plant leaf disks are performedanalogous to those described in U.S. Pat. No. 8,344,207. Anon-transformed corn plant was used to obtain tissue to be used as anegative control. Multiple transformation R₀ single-copy insertionevents from each binary vector are assessed against FAW and compared toTIC7941PL_1 to determine if insertion of the FAWPEPBIN peptide increasesthe insecticidal activity of TIC7941PL_1 against FAW.

Example 8 Reduction of TIC7941PL_1 Expression in the Reproductive Tissueof Stably Transformed Soybean Plants Through the Use of miRNA TargetSites

This example illustrates the reduction of expression of TIC7941PL_1 inthe reproductive tissues of stably transformed soybean plants throughthe use of operably linked miRNA recognition sites.

Plant miRNAs regulate target genes at the post-transcriptional level viatwo major mechanisms: transcript cleavage and translation repression. Inaddition, some miRNAs also trigger the production of secondary shortinterfering RNAs (siRNAs) from their transcripts, amplifying the effectof the miRNA on expression. miRNAs are usually twenty-one (21)nucleotides in length, but those that trigger the production ofsecondary siRNAs, are twenty-two (22) nucleotides in length. Throughdata mining of miRNAs in various tissues in soybean, two miRNAs wereidentified that were over-represented in reproductive tissues whencompared to vegetative tissues; miR395 and miR4392. miR395 is processedinto a twenty one (21) nucleotide miRNA/miRNA® duplex and is expressedmostly in the soybean flower stamen. miR4392 is processed into a twentytwo (22) nucleotide miRNA/miRNA® duplex and triggers the production ofsecondary siRNAs from its transcripts, amplifying the suppressionsignal. miR4392 is highly enriched in the soybean flower anthers. Boundwith an ARGO protein to form a silencing complex, miRNAs function assequence-specific guides, directing the silencing complex to transcriptsthrough base pairing between the miRNA and the miRNA target bindingsites within the 3′ untranslated region (3′ UTR) of the target RNAs.

Target sites corresponding to miR395 (Gm.miR395_1 (SEQ ID NO:18) andGm.miR395_2 (SEQ ID NO:19)) were operably linked using the DNA spacer(SP-ART.8a-1, SEQ ID NO:24) to construct SUP-miR395 (SEQ ID NO:20).Target sites corresponding to miR4392 (Gm.miR4392_1 (SEQ ID NO:21) andGm.miR4392_2 (SEQ ID NO:22)) were operably linked using the DNA spacer(SP-ART.8a-1, SEQ ID NO:24) to construct SUP-miR4392 (SEQ ID NO:23).SUP-miR395 and SUP-miR4392 were operably linked to the TIC7941PL_1coding sequence 3′ after the stop codon producing the transgenes,TIC7941PL_1-miR395 (SEQ ID NO:25) and TIC7941PL_1-miR4392 (SEQ IDNO:26), respectively.

Binary plant transformation vectors comprising transgene cassettesdesigned to express untargeted TIC7941PL_1-miR395 andTIC7941PL_1-miR4392 were constructed using methods known in the art andwere similar to those described in Example 4. Two constructs wereconstructed using the same promoter, leader and 3′ UTR elements asConstruct 3 in Example 4 and comprised the TIC7941PL_1-miR395 andTIC7941PL_1-miR4392 DNA sequences. Multiple transformation events fromeach binary vector were assessed using leaf discs against SAW, SBL, SPW,and VBW as described in in Example 4. Construct 3, TIC7941PL_1, servedas a control for comparison of insecticidal activity of the constructscomprising untargeted TIC7941PL_1-miR395 and TIC7941PL_1-miR4392

TABLE 6 Leaf damage ratings (LDR) and penetrance for transformed soybeanR₀ events expressing TIC7941PL_1. TIC7941 LDR (Observed/Assayed)Construct Composition SAW SBL SPW VBC Construct TIC7941PL_1 1 (12/12) 1(12/12) 1 (12/12) 3 (5/12) 3 Construct TIC7941PL_1- 1 (20/20) 1 (20/20)1 (20/20) 3 (9/20) 5 mi395 Construct TIC7941PL_1- 1 (17/19) 1 (17/19) 1(16/19) 3 (3/19) 6 mi4392

As can be seen in Table 6, operably linking miRNA target binding sitesto the TIC7941PL_1 coding sequence did not affect the insecticidalactivity of TIC7941PL_1. The two miRNA target binding site constructsdemonstrated the same level of insecticidal activity against SAW, SBL,SPW, and VBC. As previously observed in Example 4, TIC7941PL_1demonstrated high resistance with high penetrance against SAW, SBL, andSPW.

All of the compositions disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions of this invention have been describedin terms of the foregoing illustrative embodiments, it will be apparentto those of skill in the art that variations, changes, modifications,and alterations may be applied to the composition described herein,without departing from the true concept, spirit, and scope of theinvention. More specifically, it will be apparent that certain agentsthat are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope, andconcept of the invention as defined by the appended claims.

All publications and published patent documents cited in thespecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A recombinant nucleic acid molecule comprising a heterologouspromoter operably linked to a polynucleotide segment encoding apesticidal protein or pesticidal fragment thereof, wherein: a. saidpesticidal protein comprises the amino acid sequence of SEQ ID NO:4, SEQID NO:2, SEQ ID NO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ IDNO:14; or b. said pesticidal protein comprises an amino acid sequencehaving at least 80% or, 85%, or 90%, or 95%, or 98% or 99%, or about100% amino acid sequence identity to SEQ ID NO:4, SEQ ID NO:2, SEQ IDNO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14; or c.said polynucleotide segment hybridizes under stringent hybridizationconditions to a polynucleotide having the nucleotide sequence of SEQ IDNO:3, SEQ ID NO:1, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,or SEQ ID NO:13.
 2. The recombinant nucleic acid molecule of claim 1,wherein: a. said recombinant nucleic acid molecule comprises a sequencethat functions to express the pesticidal protein in a plant; or b. saidrecombinant nucleic acid molecule is expressed in a plant cell toproduce a pesticidally effective amount of the pesticidal protein orpesticidal fragment; or c. said recombinant nucleic acid molecule is inoperable linkage with a vector, and said vector is selected from thegroup consisting of a plasmid, phagemid, bacmid, cosmid, and a bacterialor yeast artificial chromosome.
 3. The recombinant nucleic acid moleculeof claim 1, defined as present within a host cell, wherein said hostcell is selected from the group consisting of a bacterial cell and aplant cell.
 4. The recombinant nucleic acid molecule of claim 3, whereinsaid bacterial host cell is from a genus of bacteria selected from thegroup consisting of: Agrobacterium, Rhizobium, Bacillus, Brevibacillus,Escherichia, Pseudomonas, Klebsiella, Pantoea, and Erwinia.
 5. Therecombinant nucleic acid molecule of claim 4, wherein said Bacillusspecies is Bacillus cereus or Bacillus thuringiensis, said Brevibacillusis Brevibacillus laterosperous, and said Escherichia is Escherichiacoli.
 6. (canceled)
 7. The recombinant nucleic acid of claim 2, whereinsaid plant cell is selected from the group consisting of an alfalfa,banana, barley, bean, broccoli, cabbage, brassica, carrot, cassava,castor, cauliflower, celery, chickpea, Chinese cabbage, citrus, coconut,coffee, corn, clover, cotton, a cucurbit, cucumber, Douglas fir,eggplant, eucalyptus, flax, garlic, grape, hops, leek, lettuce, Loblollypine, millets, melons, nut, oat, olive, onion, ornamental, palm, pasturegrass, pea, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin,Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub,sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet corn, sweet gum, sweet potato,switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon,and wheat plant cell.
 8. The recombinant nucleic acid molecule of claim1, wherein said protein exhibits activity against a Lepidopteran insect.9. The recombinant nucleic acid molecule of claim 8, wherein saidLepidopteran insect is selected from the group consisting of: Velvetbean caterpillar, Sugarcane borer, Lesser cornstalk borer, Corn earworm,Tobacco budworm, Soybean looper, Black armyworm, Southern armyworm, Fallarmyworm, Beet armyworm, American bollworm, Oriental leaf worm, Pinkbollworm, Black cutworm, Southwestern Corn Borer, Cotton leaf worm,Diamond back moth, Spotted boll worm, Tobacco cut worm, Western beancutworm and European corn borer.
 10. A plant, or part thereof,comprising the recombinant nucleic acid molecule of claim
 1. 11.(canceled)
 12. The plant of claim 10, wherein said plant is selectedfrom the group consisting of an alfalfa, banana, barley, bean, broccoli,cabbage, brassica, carrot, cassava, castor, cauliflower, celery,chickpea, Chinese cabbage, citrus, coconut, coffee, corn, clover,cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax,garlic, grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut,oat, olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper,pigeon pea, pine, potato, poplar, pumpkin, Radiata pine, radish,rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southernpine, soybean, spinach, squash, strawberry, sugar beet, sugarcane,sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea,tobacco, tomato, triticale, turf grass, watermelon, and wheat.
 13. Aseed of the plant of claim 10, wherein said seed comprises saidrecombinant nucleic acid molecule.
 14. An insect inhibitory compositioncomprising the recombinant nucleic acid molecule of claim
 1. 15. Theinsect inhibitory composition of claim 14, further comprising anucleotide sequence encoding at least one other pesticidal agent that isdifferent from said pesticidal protein.
 16. The insect inhibitorycomposition of claim 15, wherein: a. said at least one other pesticidalagent is selected from the group consisting of an insect inhibitoryprotein, an insect inhibitory dsRNA molecule, and an ancillary protein;b. said at least one other pesticidal agent exhibits activity againstone or more pest species of the orders Lepidoptera, Coleoptera, orHemiptera; or c. said at least one other pesticidal protein is selectedfrom the group consisting of a Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae,Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A/F chimeras,Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3,Cry3A variants, Cry3B, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34,Cry35, Cry43A, Cry43B, Cry51Aa1, ET29, ET33, ET34, ET35, ET66, ET70,TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC853, TIC900,TIC901, TIC1201, TIC1415, TIC3131, TIC2160, VIP3A, VIP3B, VIP3Ab,AXMI-001, AXMI-002, AXMI-030, AXMI-035, AXMI-036, AXMI-045, Axmi52,Axmi58, Axmi88, Axmi97, Axmi102, Axmi112, Axmi117, Axmi100, AXMI-115,AXMI-113, and AXMI-005, AXMI134, AXMI-150, Axmi171, AXMI-184, axmi196,axmi204, axmi207, axmi209, Axmi205, AXMI218, AXMI220, AXMI221z,AXMI222z, AXMI223z, AXMI224z and AXMI225z, AXMI238, AXMI270, AXMI279,AXMI335, AXMI345, AXMI-R1, and variants thereof, IP3 and variantsthereof, DIG-3, DIG-5, DIG-10, DIG-11, DIG-657 protein, PHI-4 variants,PIP-72 variants, PIP-45 variants, PIP-64 variants, PIP-74 variants,PIP-77 variants, DIG-305, PIP-47 variants, DIG-17, DIG-90, DIG-79, andDIG-303.
 17. (canceled)
 18. (canceled)
 19. The insect inhibitorycomposition of claim 14, defined as comprising a plant cell thatexpresses said recombinant nucleic acid molecule of claim
 1. 20. Acommodity product produced from the plant, or part thereof, of claim 10,wherein said commodity product comprises a detectable amount of saidrecombinant nucleic acid molecule or a pesticidal protein.
 21. Thecommodity product of claim 20, selected from the group consisting ofcommodity corn bagged by a grain handler, corn flakes, corn cakes, cornflour, corn meal, corn syrup, corn oil, corn silage, corn starch, corncereal, and the like, and corresponding soybean, rice, wheat, sorghum,pigeon pea, peanut, fruit, melon, and vegetable commodity productsincluding, where applicable, juices, concentrates, jams, jellies,marmalades, and other edible forms of such commodity products containinga detectable amount of such polynucleotides and or polypeptides of thisapplication, whole or processed cotton seed, cotton oil, lint, seeds andplant parts processed for feed or food, fiber, paper, biomasses, andfuel products such as fuel derived from cotton oil or pellets derivedfrom cotton gin waste, whole or processed soybean seed, soybean oil,soybean protein, soybean meal, soybean flour, soybean flakes, soybeanbran, soybean milk, soybean cheese, soybean wine, animal feed comprisingsoybean, paper comprising soybean, cream comprising soybean, soybeanbiomass, and fuel products produced using soybean plants and soybeanplant parts.
 22. A method of producing seed, the method comprising: a.planting a first seed according to claim 13; b. growing a plant orplants from said seed; and c. harvesting seed from said plant or plants,wherein said harvested seed comprises said recombinant nucleic acidmolecule.
 23. (canceled)
 24. A method for controlling a Lepidopteranspecies pest or pest infestation, said method comprising contacting thepest with a pesticidally effective amount of the pesticidal proteinencoded by the recombinant nucleic acid molecule of claim
 1. 25. Amethod of detecting the presence of the recombinant nucleic acidmolecule of claim 1 in a sample comprising plant genomic DNA,comprising: a. contacting said sample with a nucleic acid probe thathybridizes under stringent hybridization conditions with genomic DNAfrom a plant comprising the recombinant nucleic acid molecule of claim1, and does not hybridize under such hybridization conditions withgenomic DNA from an otherwise isogenic plant that does not comprise therecombinant nucleic acid molecule of claim 1, wherein said probe ishomologous or complementary to SEQ ID NO:3, SEQ ID NO: 11, or SEQ ID NO:13, or a sequence that encodes a pesticidal protein comprising an aminoacid sequence having at least 80%, or 85%, or 90%, or 95%, or 98%, or99%, or about 100% amino acid sequence identity to SEQ ID NO:4, SEQ IDNO:2, SEQ ID NO:12, or SEQ ID NO:14; b. subjecting said sample and saidprobe to stringent hybridization conditions; and c. detectinghybridization of said nucleic acid probe with said plant genomic DNA ofsaid sample.
 26. A method of detecting the presence of a pesticidalprotein encoded by the recombinant nucleic acid molecule of claim 1,comprising: a. contacting said sample with an immunoreactive antibody;and b. detecting the presence of said pesticidal protein, or fragmentthereof.
 27. The method of claim 26, wherein the step of detectingcomprises an ELISA, or a Western blot.
 28. A method for improving thepesticidal activity of a native pesticidal protein against an insectpest species, comprising: engineering a variant pesticidal protein byinserting a DNA fragment encoding an insect midgut receptor bindingpeptide into a coding sequence encoding the pesticidal protein; whereinthe pesticidal activity of the engineered pesticidal protein is greaterthan the pesticidal activity of the native pesticidal protein to saidinsect pest species.
 29. The method of claim 28, wherein: a. the insectgut receptor is selected from the group consisting of a cadherin-likeprotein (CADR), a GPI-anchored aminopeptidase-N (APN), a GPI-anchoredalkaline phosphatase, a transmembrane ABC transporter, and an ADAMmetalloprotease; b. the DNA fragment encoding an insect gut receptorbinding peptide is selected from the group consisting of SEQ ID NO:15and SEQ ID NO:16; or c. the gut receptor binding peptide is SEQ IDNO:17.
 30. (canceled)
 31. (canceled)
 32. A recombinant nucleic acidmolecule comprising a heterologous promoter operably linked to apolynucleotide segment encoding a pesticidal protein or pesticidalfragment thereof, operably linked to a DNA sequence comprising areproductive tissue-specific miRNA target binding site element, whereinsaid miRNA target binding site element is heterologous with respect tosaid polynucleotide segment encoding a pesticidal protein or pesticidalfragment thereof.
 33. The miRNA target binding site element of claim 32,selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
 34. A recombinantDNA molecule selected from the group consisting of SEQ ID NO:25 and SEQID NO:26.
 35. A method for reducing expression of a pesticidal proteinin the reproductive tissue of a transgenic plant, comprising expressingin said transgenic plant the recombinant nucleic acid molecule of claim32.